Barrier function of porcine choroid plexus epithelial cells is modulated by cAMP-dependent pathways in vitro

doi:10.1016/S0006-8993(99)02317-3

Brain Research

Volume 853, Issue 1, 17 January 2000, Pages 115-124

https://doi.org/10.1016/S0006-8993(99)02317-3 Get rights and content

Abstract

In the present paper, we demonstrate that the barrier properties of primary cultured epithelial cells isolated from porcine choroid plexus are regulated by cAMP-dependent signal transduction pathways in vitro. Triggering cAMP-connected cascades in cell layers grown on permeable filters with cAMP-analogues or forskolin led to a significant increase of transepithelial electrical resistances and a pronounced reduction in the permeation rate of a 4 kDa-dextran probe. In dose–response experiments using the cAMP-analogue 8-(4-chlorophenylthio)-cAMP transepithelial electrical resistances were observed to increase above a threshold concentration ranging between 10^−5.5 and 10⁻⁵ M. Additional impedance studies performed with confluent cell layers grown on gold-film electrodes revealed that the observed changes in transepithelial resistances and presumably also in macromolecular permeation rates were not entirely caused by a reinforcement of intercellular junctions but also contained contributions from changes in the cell-substrate adhesion pattern. These inherent contributions to the electrical resistance and macromolecular permeability are caused by a restricted diffusion pathway between basal plasma membrane and culture substrate that have to be considered in data analysis, especially when leaky cell layers on filter substrates with low pore densities are used.

Introduction

Two cellular barriers effectively protect the mammalian brain from passive entrance of ions and hydrophilic compounds circulating in the bloodstream and thereby guarantee a constant chemical environment within the central nervous system (CNS). First, the capillaries of the brain are almost entirely lined by very unique endothelial cells that build up the so-called blood–brain barrier. Second, in very limited regions of the brain, where the endothelial cells do not provide a sufficiently tight barrier — namely in certain parts of the ventricular system, the choroid plexus — an underlying sheet of epithelial cells serves to separate blood from cerebrospinal fluid (blood–cerebrospinal-fluid barrier). The ability of both cell types to separate two compartments of different chemical composition arises from the formation of very tight intercellular junctions (tight junctions) [25]. These cell–cell contacts prevent diffusive permeation of blood derived compounds along the intercellular cleft between adjacent cells into the CNS or vice versa. Besides the sole barrier function, the choroid plexus epithelial cells, which are in the focus of the present study, are responsible for a multitude of physiological needs like active transport of micronutrients into the brain, clearance of certain toxic compounds out of the brain and eventually production of cerebrospinal fluid itself [5]. The choroid plexus is particularly important from a medical viewpoint since delivery of hydrophilic drugs to the CNS via the bloodstream is hampered by the same barriers that are responsible for CNS homeostasis as described above. Clearance activity of plexus cells and the expression of `multidrug-resistance' transport proteins [22] increase the difficulties to establish and maintain effective drug concentrations in the brain capable to treat neuronal diseases. Hence, there is considerable interest to understand the signal transduction pathways that control the barrier function of this epithelium in detail.

We recently introduced an in vitro model of the blood–cerebrospinal-fluid barrier based on choroid plexus epithelial cells isolated from porcine brain 9, 13, 14. When these cells are cultured on permeable filter inserts, the apical side of the cell layer mimics the ventricular compartment, whereas the basolateral side models the blood side. We were able to show that these cells maintain their characteristic structural and functional properties in vitro 9, 14. They express microvilli, ciliae and complex tight junctions and show active transport of micronutrients, antibiotics and fluid as well as the polar secretion of proteins that are unique for the cerebrospinal fluid [9]. In the present study, we explored the impact of the cAMP signal transduction cascade on the barrier function of these cells. The cAMP-cascade seemed challenging to us, as it is well known from epithelia and endothelia of other origins, that cAMP is often involved in barrier regulation 4, 8, 24, 28. It is furthermore known that the endothelial cells in cerebral capillaries, which fulfill similar physiological functions, respond to increased cAMP levels by barrier strengthening 6, 23.

Epithelial barrier function is commonly quantified by measuring the permeation rate of a marker compound of a certain molecular weight or by the very convenient determination of transepithelial electrical resistances (TER) as a measure of ionic permeability. Although both experimental approaches are well established and widely accepted, under some circumstances data interpretation may require some caution. Recently, Lo et al. [18] pointed out that the outcome of transepithelial resistance measurements for cell layers grown on porous filters may be affected by the separation distance between the basal cell membrane and the culture substrate. The reason for this is that any paracellular current that traverses the epithelium has to flow through the pores of the filter, then through the narrow channels between basal cell membrane and substrate, before it can escape through the paracellular shunt between adjacent cells. This current pathway underneath the cells provides an additional resistance that is strongly dependent on cell-substrate separation distance. It is inherently included in TER values determined by both the common DC-techniques or AC impedance analysis as long as cells are grown on a porated filter. Although not reported before, the same is true for permeability studies using macromolecular probes that have to diffuse along the same paracellular pathways as the ions in electrochemical experiments. However, when culturing the cells on top of solid gold-film electrodes, impedance analysis can distinguish between the resistance contribution arising from intercellular contacts between adjacent cells (barrier function) and the resistance that arises from cell-substrate adhesion [10]. This technique has been pioneered by Giaever and Keese 10, 11, and it is referred to as $e$ lectric $c$ ell-substrate $i$ mpedance $s$ ensing (ECIS).

In the present study, we analyzed the influence of elevated intracellular cAMP-levels on the barrier properties of choroid plexus epithelial cells grown on porous filters by measuring both transepithelial resistances and macromolecular permeabilities. Both methods consistently implicated a distinct increase of barrier efficiency. We additionally studied the response of plexus cells to cAMP-elevation when cells were grown on gold-film electrodes. Impedance analysis and subsequent data modeling confirmed that under these conditions, plexus cells strengthen their intercellular contacts and therefore enhance their barrier function. But we also observed significant changes associated with the cell-substrate contact zone that could not have been accounted for with the commonly used filter methods.

Section snippets

Cell preparation and culture conditions

Epithelial cells from porcine choroid plexus were prepared as already described elsewhere [9]. Briefly, choroid plexus tissue taken from freshly slaughtered pigs was incubated with 0.25% (w/v) trypsin solution (Biochrom, Berlin, Germany) for 2.5 h at 4°C and then for an additional 0.5 h at 37°C. Proteolytic activity was terminated by addition of newborn calf serum (Biochrom). Enzymatically unreleased tissue was separated from the cell suspension, and the released cells were spun down at 15×g.

Results

For the present study, we made use of an in vitro model of the blood–cerebrospinal fluid barrier (CSF) that is based on primary cultured choroid plexus epithelial cells derived from pig brain 9, 13, 14. Typical values for the cell layers' permeability towards macromolecular probes are 1.5·10⁻⁸ cm/s for 150 kDa-dextran and 5.0·10⁻⁷ cm/s for 4 kDa-dextran [14]. Transepithelial resistances were usually found between 100 and 150 Ω cm². For the cell layers used here we determined average values

Discussion

The physiological regulation of epithelial and endothelial barrier function in the various organs of animal bodies has been the subject of numerous studies throughout the past years 3, 6, 8, 15, 24, 28. These investigations were mostly driven by the lack of understanding of how the cells comprising these interfacial tissues can adjust their barrier properties to particular physiological needs and also how pathological conditions manage to disrupt these anatomical barriers. Among others, the

Acknowledgements

This work has been financially supported by the German Ministry of Education and Research (BMBF) under contract No. 03114666B. The authors would like to thank Dr. I. Giaever and Dr. C.R. Keese (Rensselaer Polytechnic Institute, Troy, NY) for their support in impedance data modeling, careful revision of the manuscript and helpful discussions. A scholarship granted to J. Wegener by the Studienstiftung des Deutschen Volkes is gratefully acknowledged.

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¹: Present address: Department of Physics and Biology, School of Science, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, USA.

²: Present address: Department of Neurosurgery, Rhode Island Hospitals/Brown University, 593 Eddy Street, Providence, RI 02902, USA.

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Research reportBarrier function of porcine choroid plexus epithelial cells is modulated by cAMP-dependent pathways in vitro

Abstract

Introduction

Section snippets

Cell preparation and culture conditions

Results

Discussion

Acknowledgements

Neurosci. Lett.

Brain Res.

Biosens. Bioelectron.

J. Electroanal. Chem.

Chem. Phys. Lipids

J. Biochem. Biophys. Methods

Prostaglandins I2 and E2 have a synergistic role in rescuing epithelial barrier function in porcine ileum

J. Clin. Invest.