Functional properties of the paracellular pathway in some leaky epithelia

We here review the functional properties of the paracellular pathway of leaky epithelia such as gallbladder and renal proximal tubule. These epithelia are characterized by leaky terminal bars between adjacent cells which allow small ions, non-electrolytes and water to leak from lumen to interstitial fluid or back. In the past 10 years a great deal of information has been obtained about the properties of the misnamed 'tight' junctions in the terminal bars, by assuming that the overall permeation pattern reflected predominantly the junctional permeation properties. Although recent trans- and intraepithelial impedance analyses indicate that this assumption is not always justified (the contribution of the lateral intercellular space to the paracellular shunt resistance is not negligible, when the spaces are collapsed) it seems that the major conclusions are correct. The properties of the terminal junctions may thus be summarized as follows. (1) Large molecules such as horseradish peroxidase are not able to pass. (2) Passage of lipophilic sub-stances is insignificant, as these substances permeate by the cellular route (3). Depending on the tissue, ion permeation is either governed by channels with negative fixed charges, or positive fixed charges, or both. As inferred from ion selectivity patterns the channels of different epithelia are either wide and highly hydrated or narrow and poorly hydrated, thus allowing more or less water molecules to pass besides the ions. In narrow channels single-file diffusion may occur. (4) Besides the selective channels a free solutions hunt seems to be present in some epithelia. (5) When applied in millimolar concentrations 2, 4, 6-triaminopyrimidinium and amiloride block negatively charged junctional channels. However these substances do not simply turn leaky epithelia into tight epithelia, because they have additional effects on the cell membranes. (6) As observed in cell cultures, formation of tight junctions requires connecting particles to be present on the cell surface — which seems to be controlled by the cytoskeleton - and requires the presence of calcium ions as ligands. (7) Cellular control over paracellular permeability may be exerted through changes of intracellular calcium concentration

Rapid determination of intraepithelial resistance barriers by alternating current spectroscopy II. Test of model circuits and quantification of results

The impedance of Necturus gallbladder epithelium was measured with transepithelial and intracellular microelectrodes in different transport states. The data are analysed with five electrical equivalent circuits, which differ with respect to the configuration of the paracellular shunt path (lumped vs distributed model of the lateral space), and of the apical cell membrane (non-ideal capacitance or surface amplification by micro-tubular infoldings). Least square fits indicate: 1. that the lumped model cannot represent the epithelium properly, even under control conditions; 2. that the distributed model, which considers the lateral intracellular space separately as a cable-like structure, describes the data well, both under control conditions and during collapse of the lateral spaces; and 3. that the above indicated variations of the apical membrane configuration improve the fits, but have little effect on the magnitude of the calculated circuit parameters. Quantitatively the analysis of 214 measurements on 25 gallbladders under control conditions yields the following results: The resistances of the tight junctions, of the lateral intercellular space, and of the apical and basal cell membrane are Rj = 123, Rlis = 35.5, Ra approximately 3,500, and Rb = 225 (all in omega cm2), and the capacitances of the cell membranes are Ca = 4.95 and Cbl = 26.5 (mu F/cm2). In oxygen deficiency and after cessation of chamber perfusion transport decreased, the spaces collapsed, and Rlis increased to approximately 130 omega cm2. Although the accuracy of the estimated Ra values is still limited, the analysis shows that it is possible to determine Rj and Rlis separately and to follow their changes in response to experimental maneuvers

Electrical impedance analysis of leaky epithelia: theory, techniques, and leak artifact problems

Publisher Summary: This chapter describes the key technical concepts of the combined transepithelial and intraepithelial impedance analysis that offers a new dimension to the exploration of the intraepithelial organization of ion transport barriers. In addition, the chapter summarizes a series of experiments in which the influence of various leaks on impedance measurements is systematically investigated. The observations from the experiments explain the basis of local inhomogeneities in current density that occur in the low-frequency range and disappear in the high-frequency range when the capacitive membrane elements effectively short-circuit the epithelia. The data presented helps the experimenter in recognizing leak artifacts in measurements and finding ways to avoid those artifacts, and these observations may serve as a starting point for the development of new techniques of leak identification in a more general sense

Impedance Analysis of Necturus Gallbladder Epithelium Using Extra- and Intracellular Microelectrodes

Publisher Summary: This chapter reviews the impedance analysis of Necturus gallbladder epithelium using extra- and intracellular microelectrodes. Because epithelia consist of serial and parallel arrays of resistive barriers, it is impossible to determine the properties of an individual barrier from transepithelial measurements alone. Therefore, transepithelial alternating current spectroscopy are combined with intracellular measurements. The data are analyzed by least squares computer, which fits with model equations in order to derive the magnitude of the equivalent-circuit parameters of the cell membranes and of the shunt path. Two electrical analog circuits are tested: (1) the lumped model in which the cell membranes and the shunt path are represented by RC elements and by a single resistor, respectively and (2) the distributed model in which the resistances of the lateral cell membrane and of the lateral space fluid are distributed in the form of a cable-like structure, which extends from the tight junction to the serosal fluid compartment. Under control conditions with extended lateral spaces, both models describe the data almost equally as well. This is because the lateral space resistance is rather small. However, when the spaces are collapsed by passage of lumen-negative current the lumped model fits are no longer adequate, particularly in the high-frequency region of the impedance plots. Under those conditions, the distributed model fits indicate that the lateral space resistance can increase to 50% or more of the transepithelial resistance. These experiments allow resolving the paracellular shunt resistance into its contributions from the tight junction proper and from the lateral space

Square wave pulse analysis of cellular and paracellular conductance pathways in necturus gallbladder epithelium

In search for a rapid and reliable method to identify and quantitatively determine cell membrane resistances and paracellular shunt resistances in epithelia we have developed appropriate techniques to measure transepithelial and intracellular potential transients in response to transepithelially applied square wave constant current pulses. Model considerations indicate that in a unilayered, homogeneous epithelium with open lateral spaces the transient potential response across each cell membrane should obey a single exponential function in case the tight junction resistance is high, as in a tight epithelium, whereas in a leaky epithelium it should consist of a superposition of two exponentials with equal sign at the membrane with the higher intrinsic time constant and of two exponentials of different sign (overshoot with recline) at the membrane with the lower intrinsic time constant. The latter predictions were experimentally verified in a study on Necturus gallbladder epithelium and equivalent circuit parameters for the cell membrane resistances and capacitances as well as for the resistance of the shunt path were calculated from the data by curve fitting procedures. The resistances of the apical and basal cell membrane and of the shunt path averaged 1220, 201 and 91 Ω cm2 respectively while the apical and basal cell membrane capacitances were 8.0 and 26.3 μF/cm2 respectively. The fact that the resistance values are 4–15 times lower than estimates derived previously from 2D-cable analysis relates to a better preservation of the transport function under the present incubation conditions as verified by a new series of cable analysis data. The capacitances agree well with estimates of the surface amplification of the cell membranes from electronmicrographs, thus confirming the validity of the interpretation of the observed voltage transients