Paracellular permeability

The blood-brain barrier (BBB) forms a physiological barrier between the central nervous system and the blood circulation. It consists of glial cells and a special species of endothelial cells, which form tight junctions between each other thereby inhibiting paracellular transport. In addition, the endothelial cells of the BBB express a variety of ABC-transporters to protect the brain tissue against toxic metabolites and xenobiotics. The BBB is permeable to water, glucose, sodium chloride and non-ionised lipid-soluble molecules but large molecules such as peptides as well as many polar substances do not readily permeate the battier. 

PAMPA is typically used to make a prediction of the passive, transcellular absorption of a compound. Compounds which may be absorbed by a paracellular mechanism or may be substrates for active transport (uptake or efflux) are usually better assessed in a cell based system. A combination of assays can be applied to gain a greater understanding of the permeability and transport properties of a compound. 

In series with a desolvation energy barrier required to disrupt aqueous solute hydrogen bonds [14], the lipid bilayer offers a practically impermeable barrier to hydrophilic solutes. It follows that significant transepithelial transport of water-soluble molecules must be conducted paracellularly or mediated by solute translocation via specific integral membrane proteins (Fig. 6). Transcellular permeability of lipophilic solutes depends on their solubility in GI membrane lipids relative to their aqueous solubility. This lumped parameter, membrane permeability,... 

Figure 1 General pathways through which molecules can actively or passively cross a monolayer of cells. (A) Endocytosis of solutes and fusion of the membrane vesicle with the opposite plasma membrane in an active process called transcytosis. (B) Similar to A, but the solute associates with the membrane via specific (e.g., receptor) or nonspecific (e.g., charge) interactions. (C) Passive diffusion between the cells through the paracellular space. (C, C") Passive diffusion (C ) through the cell membranes and cytoplasm or (C") via partitioning into and lateral diffusion within the cell membrane. (D) Active or carrier-mediated transport of an otherwise poorly membrane permeable solute into and/or out of a cellular barrier.

It is assumed that the convective flow of water across the ABL, cell mono-layer, and filter owing to pressure gradients is negligible and that the cell mono-layer is uniformly confluent. When these conditions are not met, Katz and Schaeffer (1991) and Schaeffer et al. (1992) point out that mass transfer resistances of the ABL and filter [as described in Eq. (21)] cannot be used simply without exaggerating the permeability of the cell monolayer, particularly the paracellular route. An additional diffusion cell design was described by Imanidis et al. (1996). 

Table 5 Permeability Coefficients of the Paracellular Pathway and Estimation of the Effective Pore Radius and Molecular Restriction Factor for the Caco-2 Cell Monolayer... 

The plot of permeability coefficient versus molecular radius in Figure 10 shows the interdependence of molecular size and electric charge. The permeability of the solutes decreases with increasing size. The protonated amines permeate the pores faster than neutral solutes of comparable size, and the anions of weak acids permeate the pores at a slower rate. The transport behavior of the ionic permeants is consistent with a net negatively charged paracellular route. These results are phenomenologically identical to those found in the transport kinetics of... 

Figure 10 Paracellular permeability of charged solutes as a consequence of their molecular radius illustrates the mechanism of molecular restricted diffusion across negatively charged pores. Solid line depicts the curve drawn through the permeability coefficients of neutral solutes, and the neutral image of positively and negatively charged permeants.

Table 7 Permeability Coefficients of the Paracellular Route of Unperturbed and Cytochalasin D-Perturbed MDCK Cell Monolayers at 25°C... 

To estimate the relative importance of the tight junction and the lateral space composing the paracellular route, let us consider the permeability of mannitol across Caco-2 and MDCK cell monolayers. The results taken from earlier examples are presented below ... 

Table 9 Theoretical Assessment of Lateral Space and Tight Junction Contributions to the Paracellular Permeability of Mannitol in Caco-2 and MDCK Cell Monolayers3...

Correlation of Paracellular Permeability with Transmonolayer Electrical Resistance... 

In summary, the relationship between TER and solute permeability shown here and by Madara and Hecht (1989) emphasizes that these two measures of paracellular leakage are related but not directly correlated. The most obvious feature is that permeability as a function of TER is dependent upon the solute characteristics, primarily molecular size but also charge. The degree of correlation becomes worse as the molecular size of the solute increases. Consequently, the interrelationship between TER and solute permeability must be measured for each cell model before a minimum TER value can be selected as a prerequisite for flux studies. 

In Section III, emphasis was placed on flux kinetics across the cultured monolayer-filter support system where the passage of hydrophilic molecular species differing in molecular size and charge by the paracellular route was transmonolayer-controlled. In this situation, the mass transport barriers of the ABLs on the donor and receiver sides of the Transwell inserts were inconsequential, as evidenced by the lack of stirring effects on the flux kinetics. In this present section, the objective is to give quantitative insights into the permeability of the ABL as a function of hydrodynamic conditions imposed by stirring. The objective is accomplished with selected corticosteroid permeants which have been useful in rat intestinal absorption studies to demonstrate the interplay of membrane and ABL diffusional kinetics (Ho et al., 1977 Komiya et al., 1980). 

Upon taking PABL and Pe into account, the transmonolayer permeability coefficient (PM) was quantified and, in turn, delineated into its component permeabilities for the transcellular and paracellular pathways (Table 11). Specifically, Pparaceii was found as... 

The permeability of the cell monolayer consists of parallel transcellular and paracellular pathways. In passive diffusional transport, it is generally taken that uncharged molecules are capable of partitioning into the cell membrane and... 

Eparaceii = permeability of charged species (cationic or anionic) for the paracellular route [Eqs. (45) and (46)]... 

Knowing the fraction of nondissociated solutes at pH 7.4 and 6.5, one obtains the permeabilities of the various molecular species across the parallel trans-cellular and paracellular routes of the cell monolayer. Hence, restating Eq. (83) as... 

The permeability coefficient of the diffusional-bioconversion pathway can be delineated with the aid of Eq. (Ill) once the permeability coefficients of the ABL, filter support, and paracellular routes are known (Table 18). It is seen that 98% of the diester molecules passing through the cell monolayer take the intracellular route. 

Madara JL, D Barenberg, S Carlsson. (1986). Effects of cytochalasin D on occluding junctions of intestinal absorptive cells Further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity. J Cell Biol 102 2125-2136. 

There is growing evidence implicating Na+-dependent solute transporters and intracellular as well as extracellular Ca2+ in the physiological regulation of the paracellular pathway [81,203,204], Such modulation of paracellular permeability is especially important for drugs such as peptides and oligonucleotides that exhibit poor permeability characteristics across both the cornea and the conjunctiva [150,152,154,155], In addition, ion transporters such as Cl and Ca2+ channels have been implicated in macromolecular transport (see Sections IV.B.2 and IV.B.4). In the following discussion, some key ion transport processes and their possible roles in solute transport across epithelial tissues are summarized. 

The effects of D-glucose observed in vivo are not well reproduced in vitro. Madara [203] reported that cytoskeletal contraction and enhanced paracellular permeability were observed only in an in situ perfusion preparation and not in an isolated tissue preparation. Although its in vivo effect was not tested, 25 mM D-glucose, an effective concentration in the jejunum [47], failed to enhance the in vitro transport of sotalol (log PC = -0.62), atenolol (log PC = 0.16), or nadolol (log PC = 0.93) across the isolated conjunctiva [213], For a similar reason and possibly due to the absence of a Na+-glucose cotransporter in the cornea, 25 mM D-glucose was ineffective in increasing the corneal transport of these three drugs. 

UB Kompella, KJ Kim, VHL Lee. (1992). Paracellular permeability of a chloride secreting epithelium. Proc Int Symp Controlled Release 19 425-426. 

AJW Huang, SCG Tseng, R Kenyon. (1990). Paracellular permeability of corneal and conjunctival epithelia. Invest Ophthalmol Vis Sci 30 684-689. 

---