Carl J. Malanga

The Transport Barrier of Epithelia: A Comparative Study on Membrane Permeability and Charge Selectivity in the Rabbit

The transport barrier of the epithelia presents one of the major problems limiting the effective use of these tissues as alternate delivery routes for macromolecules such as peptides and proteins. In the present study, two membrane transport properties, namely, the permeability and permselectivity of the shunt pathway, were investigated and compared in various tissues including the nasal, tracheal, bronchial, buccal, rectal, vaginal, corneal, epidermal, duodenal, jejunal, ileal, and colonic epithelia. Membrane permeability was evaluated using a combined method based on electrical conductance and flux measurements of a hydrophilic fluorescent probe, 6-carboxy fluorescein (CF). Membrane permselectivity or the charge discriminating ability of the membrane was evaluated by KCl diffusion potential measurements. The results indicate that all epithelia under investigation possess a relatively high degree of permeation barrier and are highly selective for the absorption of positively charged solutes. Shunt path permeability was found to vary greatly among tissues from different epithelia, whereas membrane charge selectivity was relatively constant in these tissues. A good correlation was observed between membrane electrical conductance and steady-state flux of CF, indicating a paracellular transport of the compound. The rank order of the intrinsic membrane permeability was as follows: intestinal≈ nasal ≥ bronchial ≥ tracheal > vaginal ≥ rectal > corneal > buccal > skin. Membrane permselectivity, expressed as the ratio of transport number (positive over negative), ranges from 1.78 for the buccal to 1.33 for the rectal epithelium. These results suggest that, for effective delivery purposes, permeation enhancing methods, by either increasing tissue permeability or modifying drug-membrane charge selectivity, are generally required. The permeation data also suggest that the respiratory epithelia represent good alternate routes for drug delivery, particularly for those that are orally ineffective, i.e., due to extensive gastrointestinal tract degradation or first-pass metabolism.

Regulation of tight junction permeability by calcium mediators and cell cytoskeleton in rabbit tracheal epithelium

The present study investigates the mechanisms controlling tight junction permeability of the tracheal epithelium, with an emphasis on the regulatory role of intra- and extracellular calcium as well as the cell cytoskeleton. The tracheas were isolated from rabbits and their junctional permeability barrier was investigated in vitro by means of transepithelial electrical resistance measurements and flux measurements of the radiolabeled paracellular tracer, 14C-mannitol. The effects of intra- and extracellular calcium were studied using the calcium ionophore A 23187 and EGTA, and that of the cytoskeleton was investigated using cytochalasin B. Intracellular calcium of the tracheal epithelium was monitored microfluorometrically using the specific calcium indicator, Fura-2 AM (acetoxymethyl ester). The results indicate that the tight junction permeability of the trachea was significantly increased upon treatment with all three of the test compounds, as evidenced by a substantial decrease in transepithelial electrical resistance and an increase in transepithelial flux of 14C-mannitol. The effects of EGTA and cytochalasin B on the tight junction permeability are fully reversible upon removal of the compounds from the bathing media. On the other hand, tissues treated with the calcium ionophore demonstrate a partial or no recovery in membrane permeability, depending on the intracellular calcium levels. Moderate and transient increases in intracellular calcium caused a partial reversibility of the membrane resistance, while high and sustained intracellular calcium levels induce a complete irreversibility of the membrane resistance. These results suggest that high extracellular calcium levels and low intracellular calcium levels are required for the normal maintenance of the junctional permeability in the tracheal epithelium. Studies using cytochalasin B indicate that there is also a close relationship between the tight junctions and the organization of actin microfilaments. Alterations of these structures as well as cellular calcium levels can result in a substantial change in transepithelial permeability. Therefore compounds that affect tight junction permeability may exert their action through the calcium and cytoskeleton mechanisms.

Targeted gene delivery to alveolar macrophages via Fc receptor-mediated endocytosis

Alveolar macrophage (AM) plays important roles in lung homeostasis and pathogenesis of diseases. The study of macrophage gene function and regulation as well as its potential therapeutic intervention will require the development of vectors capable of safe and efficient transfer of DNA to the AM. In the present study, we report a new transfection system that utilizes Fc receptor-mediated endocytosis as a means to target DNA to the AM. This system employs molecular conjugates consisting of a cognate moiety, in this case IgG which recognizes the AM Fc receptor, covalently-linked to a DNA-binding moiety, such as a cationic polyamine. A Complex was formed between immunoglobulin G-polylysine conjugate (IgG-pL) and plasmid DNA carrying the LacZ reporter gene (pSVβ). The conjugate-DNA complex was added directly to the AMs in culture and incubated for 24 h, after which LacZ gene expression was analyzed for β-galactosidase activity by microfluorometry using a fluorogenic β-galactosidase substrate, 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG). The AMs treated with the IgG-pL/DNA complex exhibited galactosidase activity significantly augmented over background levels. Effective gene transfer was shown to require both the DNA-binding moiety and cognate moiety for the cell surface receptor. Specific internalization of the complex by the Fc receptor pathway was verified by competitive inhibition using excess IgG. Under this condition, LacZ gene expression was inhibited, suggesting complex internalization through the Fc mediated endocytosis pathway. The requirement of Fc receptors for complex internalization was further demonstrated using cells that lack Fc receptors, e.g., alveolar epithelial cells. When exposed to the IgG-pL/pSVβ complex, these epithelial cells showed no susceptibility to gene transfer. Thus, the immune conjugate system may be used to accomplish targeted gene delivery to the AMs via the endocytosis pathway. Finally, the conjugate system was found to be nontoxic at concentrations effectively enhancing gene transfer, thereby, suggesting its potential safety in vivo.

Receptor-Mediated Peptide Delivery in Pulmonary Epithelial Monolayers

The present study investigated the feasibility of utilizing receptor-mediated endocytosis as a means to enhance peptide delivery to the pulmonary epithelium. The strategy employs a molecular conjugate consisting of a cognate moiety, transferrin (TF), covalently-linked to a model polypeptide, horseradish peroxidase (HRP), via a reversible disulfide linkage. A cultured alveolar epithelial monolayer system was used to simulate the conditions of the pulmonary epithelium and to allow accurate quantitation of intra- and transcellular peroxidase transport. The alveolar cells were isolated from rat lungs by enzymatic digestion and grown on microporous tissue culture-treated polycarbonate filters. A significant increase in the uptake of HRP by the cell monolayer was observed upon its conjugation with TF. The effect was found to be concentration-dependent, being more pronounced at low concentrations, i.e., 3.9- and 1.2-fold increase over unconjugated HRP controls at the concentration levels of 0.05 and 1.50 U/ml respectively. Effective peroxidase uptake was shown to require the TF cognate moiety for the cell surface receptor. Specific internalization of the conjugate by the TF endocytic pathway was verified by competition for the TF receptor. Conjugate internalization was not followed by a proportional increase in transcytosis, i.e., at 0.05 U/ml conjugate level, a 1.7-fold increase in transcytosis was observed as compared to 3.9-fold for endocytosis. Effective enhancement of transcytosis was achieved by treating the monolayers with brefeldin A (BFA), a compound known to affect intracellular transport of TF receptor complexes. At 1.6 µ/ml concentration level, BFA promoted a >20-fold increase in the rate of transcytosis of the conjugate in both the apical-to-basal and basal-to-apical directions. This effect was not associated with membrane leakage since BFA-treated monolayers maintained tight barrier to transport of the paracellular permeability solute 14C mannitol. In addition, BFA had no significant effect on the transport of free HRP. Instead, the effect of BFA on conjugate transport was mediated by TF receptors since excess free TF competitively inhibited transcytosis of the conjugate. Thus, our results are consistent with the TF receptor-mediated transport of the conjugate and its enhancement through the intracellular rerouting of the conjugate by BFA. The findings in this study may potentially be relevant to the design of drug delivery systems that can enhance intra- or transcellular uptake of therapeutic peptides in the pulmonary epithelium.

Endogenous dopamine in bivalve gills

Abstract 1. 1. Dopamine was identified in gill extracts of Modiolus demissus, Mytilus edulis and Modiolus modulus in concentrations ranging from 0·12 to 0·98 μg/g. 2. 2. Incubation of gills in 2 × 10 −4 M l -dopa for 2 hr resulted in a greater than tenfold increase in endogenous dopamine levels. 3. 3. Norepinephrine is apparently not present at levels comparable to those of dopamine.

Alveolar Permeability Enhancement by Oleic Acid and Related Fatty Acids: Evidence for a Calcium-Dependent Mechanism

Pulmonary exposure to oleic acid (OA) is associated with permeability alterations and cellular damage; however, the exact relationship between these two effects has not been clearly established. Using cultured alveolar epithelial monolayers, we demonstrated that OA and some other fatty acids (≤50 µM) can induce permeability changes without detectable cellular damage. At higher concentrations, however, OA caused severe membrane damage and leakage to solute flux. The permeability enhancing effect of OA was observed with both the paracellular marker 3H-mannitol and the lipophilic transcellular indicator 14C-progesterone. While the effect of OA on transcellular permeability may be attributed to its known effect on membrane fluidity, the paracellular promoting effect of OA and its mechanism are not well established. We postulated that OA may increase paracellular permeability through a Ca2+-dependent tight junction mechanism. Using dual-excitation fluorescence microscopy, we demonstrated that OA can increase intracellular calcium, [Ca2+]i , in a dose-dependent manner. This effect was transient at low OA concentrations (≤50 µM) but became more pronounced and sustained at higher concentrations. Free hydroxyl and unsaturated groups were required for this activation since esterified OA (oleic methyl ester) and stearic acid (a saturated fatty acid with equal chain length) had much reduced effects on both the [Ca2+]i and the permeability alterations. Degree of unsaturation was unimportant since linolenic acid (18:3), linoleic acid (18:2), and OA (18:1) had similar and comparable effects on the two parameters. When the alveolar epithelium was bathed with Ca2+ -free medium, OA failed to elevate [Ca2+ ]i , suggesting that Ca2+ influx from the extracellular medium is responsible for the observed [Ca2+]i rise. This effect of OA was not due to nonspecific membrane damage since the monolayer maintained its integrity and the [Ca2+]ireturned to pretreatment levels after an initial transient rise. Moreover, the permeability alteration was fully reversible upon removal of OA. These results suggest that the alveolar permeability may be reversibly enhanced by sublethal concentrations of oleic acid.

Dopaminergic stimulation of frontal ciliary activity in the gill of Mytilus edulis

Abstract 1. 1. Dopamine (DA) produced a graded stimulation of the rate of crawling and particle transport by Mytilus edulis gill pieces through a mechanism proposed to involve frontal cilioexcitation. 2. 2. Epinine mimicked the graded dopaminergic stimulation, but norepinephrine, epinephrine, isoproterenol, tyramine and apomorphine did not significantly stimulate crawling rates. 3. 3. The dopaminergic stimulation of crawling was most effectively antagonized by ergonovine, 2-BOL and cyproheptadine and to a lesser extent by haloperidol and phenoxybenzamine. Methysergide and phentolamine were ineffective as antagonists. 4. 4. Even though 10 −5 M 5HT inhibited the crawling rate through a mechanism believed to be related to lateral cilioexcitation and not by inhibition of frontal cilia, 5HT effectively reduced the dopaminergic stimulation of crawling. 5. 5. Frontal ciliary activity and filter feeding may be controlled by a receptor responsive to both DA and 5HT.

Effects of the cilioexcitatory neurohumors dopamine and 5-hydroxytryptamine on cyclic AMP levels in the gill of the mussel mytilus edulis

Abstract Cyclic 3′, 5′-adenosine monophosphate (cAMP) has been identified in the ciliated gill epithelium of the marine mussel Mytilus edulis . In concentrations which stimulate the rate of particle transport by frontal gill cilia, DA and 5HT stimulate levels of cAMP within the gill. The stimulation occurs in as early as 15 sec and is graded from 10−6M to 10−4M. DA plus 5HT is not additive at maximal effective concentrations of both amines. ACH does not mimic the DA or 5HT stimulation of cAMP. Theophylline alone has a weak effect on cAMP levels; however, the effect of theophylline is potentiated in the presence of DA or 5HT. Dibutyryl cAMP produces a gradual stimulation in the rate of particle transport. It is suggested that the dopaminergic and serotonergic excitatory control of particle transport by frontal gill cilia of Mytilus edulis is mediated through a cAMP second messenger system.

Characterization of proteolytic activities of pulmonary alveolar epithelium

Pulmonary alveolar type I epithelial cell and its progenitor, type II cell, present major transport and enzyme barriers for systemic delivery of pulmonary administered peptide drugs. The present study investigates the effect of cellular differentiation of type II to type I cells on their proteolytic activities, and evaluates the suitability of a continuous lung cell line, A549, for drug transport and degradation studies. High performance liquid chromatography was used to assess the degradation kinetics of two model peptide substrates, luteinizing hormone releasing hormone (LHRH) and [D-Ala(6)10-fold decrease in proteolytic activities for LHRH, as compared to type II cells. The continuous lung cell line A549 formed leaky monolayers and exhibited similar enzyme activities to the primary type II cells. The responsible enzymes for degradation of LHRH in type II and A549 cells were angiotensin converting enzyme (ACE), EP24.11, and EP24.15. In contrast, no EP24.15 or ACE activity was observed in type I-like pneumocytes and only a weak EP24.11 activity was detected. In all cell types, the degradation rate of [D-Ala(6)]-LHRH was about 3-8 times lower than that of LHRH. This peptide analog was resistant to degradation by EP24.15 and EP24.11, but was susceptible to ACE-mediated cleavage.

Effects of dopamine on anaerobic metabolism and ciliary activity in bivalve gills

Abstract 1. Manometric measurements show that 1 × 10−4 M dopamine (DA) stimulates the rate of anaerobic glycolysis in the gills of Modiolus demissus, Mytilus edulis, and Modiolus modiolus. 2. The stimulation of glycolysis in M. demissus gill is mimicked, to a lesser extent, by l -dopa. 3. Dopamine, in concentrations from 1 × 10−7 M to 10−4 M, inhibits lateral ciliary activity, measured stroboscopically, in M. demissus gill. 4. Dopamine and l -dopa (1 × 10−4 M) completely, but reversibly, stop metachronal wave activity in M. demissus. 5. Crawling rates of Modiolus and Mytilus gill pieces are stimulated by dopamine (1 × 10−4 M), apparently by stimulation of frontal ciliary activity.