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Advanced Drug Delivery Reviews 49 (2001) 281–300www.elsevier.com/ locate /drugdeliv

Caveolae as potential macromolecule trafficking compartmentswithin alveolar epithelium

*Mark Gumbleton

Pharmaceutical Cell Biology, Welsh School of Pharmacy, Redwood Building, Cardiff University, Cardiff CF10 3XF, UK

Received 11 January 2001; accepted 3 April 2001

Abstract

With inhalational delivery the alveolar epithelium appears to be the appropriate lung surface to target for the systemicdelivery of macromolecules, such as therapeutic proteins. The existence of a high numerical density of smooth-coated ornon-coated plasma membrane vesicles or invaginations within the alveolar epithelial type I cell has long been recognised.The putative function of these vesicles in macromolecule transport remains the focus of research in both pulmonaryphysiology and pharmaceutical science disciplines. These vesicles, or subpopulations thereof, have been shown tobiochemically possess caveolin, a marker protein for caveolae. This review considers the morphometric and biochemicalstudies that have progressed the characterisation of the vesicle populations within alveolar type I epithelium. Parallelresearch findings from the endothelial literature have been considered to contrast the state of progress of caveolae research inalveolar epithelium. Speculation is made on a model of caveolae vesicle-mediated transport that may satisfy some of thepulmonary pharmacokinetic data that has been generated for macromolecule absorption. The putative transport function ofcaveolae within alveolar epithelium is reviewed with respect to in-situ tracer studies conducted within the alveolar airspace.Finally, the functional characterisation of in-vitro alveolar epithelial cell cultures is considered with respect to the role ofcaveolae in macromolecule transport. A potentially significant role for alveolar caveolae in mediating the alveolar airspace toblood transport of macromolecules cannot be dismissed. Considerable research is required, however, to address this issue ina quantitative manner. A better understanding of the membrane dynamics of caveolae in alveolar epithelium will help resolvethe function of these vesicular compartments and may lead to the development of more specific drug targeting approaches forpromoting pulmonary drug delivery. 2001 Elsevier Science B.V. All rights reserved.

Keywords: Caveolin; Caveolae; Lung; Alveolar epithelium; Transport; Endocytosis and transcytosis

Contents

1. Introduction ............................................................................................................................................................................ 2822. Caveolae and the structural role of caveolins ............................................................................................................................. 2823. Vesicular system in alveolar epithelium..................................................................................................................................... 283

3.1. Alveolar epithelial–pulmonary capillary barrier.................................................................................................................. 2833.2. Membrane vesicles in alveolar epithelium.......................................................................................................................... 2853.3. Caveolae and caveolin in alveolar epithelium ..................................................................................................................... 286

*Corresponding author. Tel. / fax: 1 44-29-2087-5449.E-mail address: [email protected] (M. Gumbleton).

0169-409X/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0169-409X( 01 )00142-9

282 M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 –300

4. Transport role for caveolae in alveolar epithelium...................................................................................................................... 2874.1. Endothelial paradigm ....................................................................................................................................................... 2884.2. In-vivo kinetic considerations and vesicular transport ......................................................................................................... 2894.3. In-situ tracer studies in alveolar airspace ............................................................................................................................ 2924.4. Caveolae and receptor-mediated transport .......................................................................................................................... 295

5. Caveolae and caveolin in cultured alveolar epithelium ............................................................................................................... 2956. Conclusion ............................................................................................................................................................................. 297References .................................................................................................................................................................................. 297

1. Introduction most frequently observed as ‘‘smooth coated’’ or‘‘non-coated’’ omega-shaped invaginations (diameter

The permeability characteristics of the lung and of 50–100 nm at the widest point) connected to therecent advances in inhalational aerosol device tech- plasmalemma or plasma membrane by a neck-likenology have led to an increasing interest in exploit- structure which affords spatial continuity with theing the pulmonary route for the systemic delivery of extracellular environment (Fig. 1a). At least inmacromolecule therapeutics, particularly recombi- endothelial cells caveolae-like vesicles may also benant proteins and polypeptides [1]. The lung deposi- observed as fused lines or clusters of vesicles at thetion and absorption studies of Colthorpe et al. [2,3] plasmalemma [7]. The term ‘‘smooth coated’’ orelegantly demonstrated that the extents of systemic ‘‘non-coated’’ vesicles has long been used to contrastabsorption of insulin or growth hormone following them with the electron-dense cytoplasmic coat thatlung administration positively correlate with the can be seen associated with clathrin-coated pits whendepth of deposition of these administered proteins viewed under the electron microscope. However, it iswithin the lung. Anatomical determinants [4] would probable that, both within a single cell and betweenalso support the view that the lung periphery, and the cell types, that various subpopulations of smooth-alveolar epithelium in particular, is the appropriate coated or non-coated vesicles exist, and not all willlung surface to target when aiming to systemically be caveolae as defined by the presence of the markerdeliver macromolecules. Some of these anatomical protein caveolin (see later).determinants would include: location of the alveolar Caveolae or caveolae-like structures are recog-surface beyond the clearance mechanisms of the nised as prominent morphological features in amucociliary escalator; the large alveolar surface area variety of cell types, notably adipocytes, muscle cellspotentially available for absorption; the high blood (skeletal, cardiac and smooth), fibroblasts, capillaryflow to the alveolar region, and the thin cellular endothelium and type I alveolar epithelial cells,barrier from airspace to capillary blood presented by although to varying extents many other cell typesthe alveolar epithelial and pulmonary capillary cells. may possess these morphological structures. AFurther, in the transport of macromolecules across principal component constituting the striated coat ofthe pulmonary alveolar epithelial–capillary endo- caveolae, and a critical structural and functionalthelial barrier, evidence indicates that it is the element of caveolae, is the cytoplasmically orien-alveolar epithelium that possesses a more restrictive tated integral membrane protein, caveolin [8]. As aparacellular pathway than that provided by the biochemical marker caveolin has provided for ancapillary endothelium [5]. As a corollary the mecha- additional definition for caveolae beyond that mor-nisms of transport of macromolecules within alveolar phological identification alone, i.e. caveolae as flat-epithelium are the subject of genuine interest [6], and tened caveolin-rich membrane microdomains mor-in particular the nature and extent of any vesicular phologically indistinguishable from the plasmalem-trafficking mechanism(s) such as that potentially ma itself (Fig. 1b).provided by caveolae. Caveolin comprises a family of proteins the most

studied of which is caveolin-1. Caveolin-1 appears tobe a critical, but not necessarily the sole, determining

2. Caveolae and the structural role of caveolins factor in caveolae formation in non-muscle cells[9–12], with the structural unit for the protein within

At the electron-microscopic level caveolae are the plasma membrane in the form of high molecular

M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 –300 283

Fig. 1. (a) Electron micrograph of a ‘flask-shaped’ caveolae invagination (CV) of diameter approximately 80 nm located within the apicalplasmalemma (PL). The invagination lacks an electron dense cytoplasmic coat characteristic of a clathrin coated pit. At the neck of thecaveolae can be seen a membraneous diaphragm (D) restricting the caveolae opening to 20–40 nm. (b) A schematic representation ofputative caveolae dynamics. Flattened caveolin-rich domains (A) may exist in the plasma membrane that steadily increase in curvature (B⇒ C) following appropriate physiological stimuli. The resultant invagination may remain attached to the plasma membrane or possiblycompletely close and detach to form a discrete intracellular vesicle.

weight caveolin oligomers [13–15]. The relationship themselves which represent the main location forbetween caveolin-1 expression and caveolae bio- gaseous exchange. Alveolar epithelium is predomi-genesis appears to require a threshold level of nantly comprised of two cell types, the terminallycaveolin expression for the formation of caveolae differentiated squamous alveolar epithelial type I[10,16], such that caveolin expression alone does not (ATI) cell which constitutes approximately 93% ofnecessarily imply the presence of caveolae within a the alveolar epithelial surface area (33% of alveolarcell. epithelial cells by number) and the surfactant produc-

ing cuboidal alveolar epithelial type II (ATII) cellcomprising the remaining 7% by surface area and

3. Vesicular system in alveolar epithelium 67% by epithelial cell number [17]. The total alveo-lar epithelial surface area within an average adult

23.1. Alveolar epithelial–pulmonary capillary human lung is estimated to be 100–120 m , althoughbarrier potentially not all of this relatively large surface is

likely to be concurrently available for the absorptionThe lower respiratory tract consists of the respira- of inhaled drug.

tory bronchioles, the alveolar ducts, and the alveoli The alveolar epithelial–pulmonary capillary bar-



rier comprises the alveolar epithelium and pulmonary 3.2. Membrane vesicles in alveolar epitheliumcapillary endothelium. In parts of the barrier thebasal membranes of the epithelial and endothelial The existence of a high numerical density ofcells are directly in contact while in other parts they membrane vesicles within the alveolar epithelial typeseparated by interstitium (Fig. 2c). Morphometric I cell has long been recognised. Morphometric datareports [17] indicate that the human ATI cell has an [19–22] obtained in the early 1980s provided im-abluminal or interstitial membrane surface area portant information on the vesicle populations within

2averaging 5098 m , with an average cell thickness of the in-vivo ATI cell. However, while the vesicle0.36 m ranging from 2 to 3 m in the perinuclear populations studied in these early investigationsregion of the cell to approximately 0.2 m in the encompassed to a high extent smooth- or non-coatedperipheral attenuated regions of the cell. Charac- vesicle populations whose morphology is consistentteristically the ATI cell displays only sparse cellular with caveolae structures, ultrastructural appearanceorganelles with the majority that are present, located alone may not be sufficient to functionally definein the cell’s perinuclear region. Some of these them as caveolae; the latter necessitating at leastmorphometric features of the ATI cell exemplify the structural association of the vesicle membrane with‘favourable’ anatomical determinants that have caveolin protein. Therefore in the following descrip-driven interest in the alveolar type I epithelium as a tion (below) of these early studies care has beenbarrier across which to deliver systemically active taken to avoid defining the vesicles explicitly asproteins and peptides. caveolae.

In constrast, the cuboidal ATII cell is considerably The studies of Gil and co-workers [19,20] de-smaller than the ATI cell (e.g. basal surface area scribed the number and distribution of plasmalemmal

2averaging 183 m and a uniform cell thickness of vesicles or invaginations within the ATI cell andabout 5 m [17]) and is richly endowed with organel- pulmonary capillary endothelial cell of rabbit lung.les and microvilli on it’s apical membrane. Current These workers did not distinguish between differentevidence would support a role for the ATII cell types of vesicles but described the presence of highserving as the sole in-vivo progenitor for, and numbers of non-coated vesicles or invaginationsdifferentiating into, the terminally differentiated ATI possessing an average diameter of 70 nm, and incell (reviewed in [18]). many cases retaining a neck-like connection to the

The pulmonary capillary endothelial cell possesses plasmalemma. The investigators did not count vesi-a very similar thin attenuated squamous morphology cles that appeared as free discrete entities within theto the ATI cell, although it’s cell surface area is cytoplasm of the cell with no apparent connection toreported to be up to three to four times smaller, e.g. either plasmalemmal surface.

2luminal surface area averaging 1353 m [17]. For the ATI cell they reported the presence of 150

Fig. 2. (a) A gallery of 19 optical images taken at steps of 0.46 m through a paraffin section of rat lung tissue using confocal laser scanningmicroscopy (CLSM). The rat lung tissue was immunostained with anti-caveolin-1 antibody and immunocolloidal gold. The colloidal goldwas visually enhanced by silver development. The very reflective particles of dense gold /silver caveolin-1 immunostain are shown in black.The images are shown in the grid in order left to right, top to bottom, starting from the uppermost surface of the paraffin section and movingthrough to the bottom of the section. Caveolin-1 staining can be seen along the surfaces of the capillary endothelium and alveolarepithelium. (As – alveolar airspace; C – capillary lumen). (b) A red/green 3-dimensional reconstruction of the 19 optical sections shown in(2a) above. A transmission image of the bright-field view of the original section has been inverted and ghosted over the 3-dimensionalreconstruction to provide a background of lung architecture. The nuclei of the cells are shown overlaid in light grey. The caveolin-1immunostaining is shown as red /green pairs. The image when viewed through red /green stereo glasses shows the 3-dimensional structure ofthe original 10-m thick paraffin section with the profile of caveolin-1 stain. (c) and (d) Transmission electron micrographs of resin-embeddedlung tissue (As – alveolar airspace; C – capillary lumen; S – surfactant). (c) Araldite thin section of tissue postfixed in osmium showing thealveolar–pulmonary capillary barrier in rat lung. Micrograph shows flask-shaped plasmalemmal invaginations or vesicles in both capillaryendothelium (right-hand surface) and alveolar type I epithelium (left-hand surface); (d) LR White thin section (osmium omitted to retainantigenicity) immunolabelled for caveolin-1. An attenuated region similar to that in (c) shows anti-caveolin-1 colloidal-gold particlesassociated with plasmalemmal invaginations in both the alveolar epithelial and capillary endothelial cells. The epithelial surface is identifiedby the presence of surfactant (S).


2vesicles per m of luminal or airspace membrane cal level. In none of the early lung alveolar mor-surface area, and the reproducible presence of a phometric papers described above were micrographs

2significantly higher vesicle load (230 vesicles per m presented, or data discussed, pertaining to the pres-cell membrane) at the basal or interstitial membrane ence of electron-dense-coated pits within the in-vivosurface. They also determined a vesicle density in ATI cell. Atwal and coworkers in their electron

3the ATI cell of 145 per m cell volume and calcu- tracer studies [27] in goat lung alluded to the lack oflated that approximately 70% of the total plasmalem- clathrin-coated pits in alveolar type I epithelium.ma surface area of the ATI cell was located within Further, in our own experience of the electron-micro-the membranes of the non-coated plasmalemmal scopic analysis of rat and human alveolar type Iinvaginations. Between species the dimensions of the epithelium we have failed to identify membraneATI cell appear similar [17,23], and based upon a structures within the ATI cell conforming to theconservative estimate of ATI cell surface area for the morphological characteristics of clathrin-coated pits.rabbit, the data of Gil et al. [19,20] would equate to However, an isolated immunoelectron microscopyapproximately 600,000 plasmalemmal vesicles or report in 1989 [28] localised clathrin light and heavyinvaginations on the luminal membrane of the ATI chains to the smooth coated vesicles seen in the typecell, and in excess of 900,000 on the abluminal I alveolar epithelial cell. These workers did notmembrane surface; statistics close to that calculated report the morphological presence of clathrin coatedby the original investigator [20]. Morphometric pits in these cells but hypothesised that some com-studies in dogs by DeFouw and colleagues [21,22] ponents that participate in clathrin-coated pits mayhave similarly reported high vesicle densities (vesicle also be involved in the formation of other vesiclediameters 48–60 nm) in the ATI cell with the types. This work has not been further substantiated.

3number of vesicles per m cell volume reported to The alveolar type I epithelium therefore appears toaverage 227–291. In the work of DeFouw [21], parallel the endothelial microvasculature in that thehowever, a significant differential distribution of majority (but not exclusively all) of the vesiclesvesicles between the luminal and interstitial mem- present in the cell are the smaller non-coated orbrane surfaces in the ATI cell was not observed. smooth-coated vesicle populations that morphologi-

At first consideration the above statistics for the cally are recognised as caveolae.ATI cell may appear extreme. Nevertheless, highnumbers of non-coated vesicles or invaginationsmorphologically conforming to caveolae are a con- 3.3. Caveolae and caveolin in alveolar epitheliumsistent feature within endothelial microvascular cells.Reports from a number of laboratories [22,24–26] With the discovery of caveolin protein, ultrastruc-each analysing different tissue beds, indicates the tural appearance alone is no longer sufficient tonumerical density of such vesicles in capillary functionally define smooth-coated or non-coated

3endothelial cells to range from 150 to 600 per m cell plasmalemmal vesicles as caveolae.volume. Indeed in the above lung morphometric In 1994 Lisanti et al. [29] described an associationstudies of Gil and coworkers [19,20], data for the between caveolin protein expression and the lung.rabbit pulmonary capillary endothelial cell was also They isolated caveolin rich domains from whole lungpresented, with vesicle numbers reported at 131 per homogenates by the use of sucrose density gradients.

3m endothelial cell volume, and vesicle loads upon Based upon ultrastructural evidence and theoretical

2each membrane of 196 per m for the luminal surface calculations they concluded that 80% of the caveolin2and 181 per m for the abluminal surface. For the signal generated by Western blot analysis is con-

smaller endothelial cell the statistics of Gil and tributed by the ATI cell. However, given the range ofcoworkers [19,20] would equate to approximately cell types that display caveolae in lung periphery200,000 plasmalemmal vesicles or invaginations on (e.g. capillary endothelial, fibroblast and alveolareach of the luminal and abluminal pulmonary capil- type I cells) coupled with the architectural complexi-lary endothelial cell membranes. ty and structural diversity of the lung tissue, the

Clathrin-coated membrane pits or invaginations calculation is tenuous.are generally readily characterised at the morphologi- The work of Kasper et al. in 1998 [30] was the


first full publication to spatially localise caveolin to The biochemical confirmation that the plasmalem-alveolar type I epithelium. Using double immuno- mal invaginations or vesicles in the in vivo ATI cellfluorescence with both frozen and paraffin wax are caveolae has a number of implications. From asections these workers reported the localisation of pharmaceutical perspective it provides a biologicalcaveolin-1 in alveolar type I epithelium of rats and framework for studies addressing the mechanisticmini-pigs. In these studies the type II alveolar role caveolae vesicles may fulfil in the trafficking ofepithelial cells were found to be devoid of caveolin-1 therapeutic macromolecules across the alveolar–staining. Following X-ray irradiation of these ani- capillary barrier, and indeed the development ofmals to induce lung injury, with subsequent initiation targeting strategies that could exploit caveolae mem-of lung fibrogenesis, Kasper and colleagues noted a brane domains for receptor mediated transcytosisdramatic loss of caveolin-1 expression in the alveolar (see article by Jan Schnitzer in this series). From aepithelium but increases in expression of caveolin-1 more fundamental basis it should provide insightsin the pulmonary capillary endothelium. They pro- into the potential regulation of endogenous soluteposed that caveolin-1 may serve as an early indicator trafficking and cell signal regulation within theof subcellular alteration during the initial stages of alveolar region. The work of Newman et al. [31] andlung fibrosis. Kasper et al. [30] has provided some initial charac-

In 1999 Newman et al. [31] undertook an im- terisation of ATI cell plasmalemma vesicles uponmunocytochemical study for caveolin in the alveolar which their transport role can be further studied withepithelial–pulmonary capillary barrier of rat lung. At reference to established functions of caveolae inthe light microscopic level they used a combination other cell types.of bright-field and confocal laser scanning micro-scopy spatially localise in a 3-dimensional mannercaveolin-1 immunomarker to alveolar epithelial andpulmonary capillary surfaces of lung tissue (Fig. 2a 4. Transport role for caveolae in alveolarand b). At the electron microscopic level they epitheliumreported observing a greater number of caveolae-likestructures in the capillary endothelium compared to While the morphometric data for the ATI cellthat seen in the ATI epithelium (Fig. 2c), although vesicle populations is intriguing in terms of theirno quantitative morphometric analyses were under- putative function as endocytic or transcytotic com-taken. These workers also noted the absence of partments, direct evidence for their role in transportcaveolae-like structures in the ATII cell. At the within alveolar epithelium is at present extremelyimmunoelectron microscopic level, however, specific limited. Certainly, the functional characterisation oflow-level labelling of the ATII cell for caveolin-1 ATI vesicle populations lags considerably behind thewas observed. Both the ATI epithelium and pulmon- progress made in determining a transport role forary capillary endothelium were specifically labelled caveolae in endothelium. However, until compara-with anti-caveolin-1 immunogold particles, with tively recently the role of endothelial caveolae inimmunogold particle frequency generally greater in vesicular trafficking events was itself much debated,the endothelium than epithelium. In both cell types due in part to the lack of recognised inhibitorsplasmalemmal invaginations could be observed deco- specific for caveolae mediated pathways, the lack ofrated with immunogold label, although not all in- ligands specifically targeting caveolae membranes,vaginations were labelled in such a manner (Fig. 2d). and also a paucity in knowledge of the underlyingThis labelling of some, but not all plasmalemmal mechanisms modulating caveolae membrane vesiclevesicles (despite their morphological similarity) was dynamics. A fuller discussion on the controversyconsidered to reflect either a true biochemical hetero- relating to the role of caveolae in endothelial trans-geneity in the smooth-coated vesicle populations port is provided in Jan Schnitzer’s article within thiswithin the ATI cell, or an antigen threshold require- series. However, to constrast the status of caveolaement for caveolin-1 coupled with a variability in the transport research in alveolar epithelium to progresslevel of this protein (or of epitope access) between that has been made in the endothelial literature, acaveolae structures. brief overview of the latter is provided below.


4.1. Endothelial paradigm in-situ studies by Palade and coworkers demon-strated the transcytosis of labelled albumin andorosomucoid (tracers that qualify as large and smallUltrastructural studies by Bungaard et al. [32,33]pore probes, respectively) via endothelial caveolae.and Frøkjaer-Jensen et al. [34,35] undertaking ul-They hypothesised that caveolae could act as largetrathin serial sectioning with reconstruction revealedpores when fully opened, and as small pores whenendothelial caveolae to be organised as fused lines orthe neck of the plasma membrane invagination isclusters of vesicles continuous with either the lumi-constricted to less than 10 nm in diameter. Converse-nal or abluminal plasmalemma. They found a paucityly, caveolae could fulfil such structural equivalentsof free discrete vesicles in the cytoplasm, and arguedthrough the presence of functionally distinct caveolaeas a result that caveolae are merely static invagina-subpopulations with a cell.tions of the cell surface and not dynamic structures

Using both cultured endothelial monolayers and ancapable of mediating endocytosis or transcytosis.in-situ model of rat lung pulmonary endothelialThis work raised the level of debate about themicrovasculature, Schnitzer and co-workers [40–42]trafficking function of endothelial caveolae, but diddemonstrated a role for caveolae-mediated transportnot provide absolute evidence against such a role,in the endothelial permeability to albumin. A specificeither via the vectorial shuttling of single discretebinding protein (originally known as gp-60 andvesicles carrying solute from one plasma membranerenamed albondin) has been identified on endothelialsurface to another, or with solute transfer mediatedsurfaces, and appears to mediate the transcytosis ofvia transient interconnections or fusions occurringnative albumin, while other endothelial surface bind-between lines or clusters of adjacent vesicles asing proteins (gp-18 and gp-30) appear to mediate thedescribed by Charles Michel [7,36].endocytosis of modified albumins [42]. Schnitzer andDuring recent years a growing body of experimen-coworkers [40] used the sterol binding agent, filipin,tal data has led to the general consensus that theto disassemble caveolae invaginations leading to aendothelial vesicle system can mediate the trans-reduction in their surface density to less than 15% ofendothelial transport of macromolecules. Of par-control, but without effect on the structural integrityticular note in this context is the work from Georgeof coated pits. Correspondingly, filipin inhibited, in aPalade’s laboratory [37–39] and that from the lab-concentration-dependent manner up to 60% of theoratory of Jan Schnitzer [40–45].transendothelial transport of native albumin acrossThe use of electron dense tracer probes to localiseboth in-vitro cultured endothelial monolayers andmacromolecules to plasma membrane vesicles suchin-situ rat lung capillaries. Fillipin treatment ex-as caveolae, and deduce from this a functionalhibited no effect upon paracellular transport path-transport role for the vesicles is impaired by aways as indicated by a lack of effect upon inulinnumber of factors. For example, artifacts may bepermeability, or indeed upon the transport of a -introduced during processing of the tissue for micro- 2

macroglobulin, a substrate internalised via clathrin-scopic analysis, or by failing to account for thecoated pits. Further, these workers showed [41] N-three-dimensional structure of a cell’s tubulo-vesicu-ethylmaleimide (NEM), an inhibitor of NEM-sensi-lar system. Work by Predescu and Palade during the

11990s [37–39], however, exploited a combination of tive factor (NSF) to reduce the endothelial transportlabelled tracer probes, quantitative permeability in-vestigations, and application of vesicular transport 1NSF is a key component in a group of proteins collectivelyinhibitors to identify that caveolae could serve as the termed the SNARE complex [46] involved in the subcellularstructural equivalents of both small and large pores trafficking and membrane fusion of vesicles. Certain componentsin continuous microvascular endothelium. For intact of the SNARE machinery, namely SNAP-25 and syntaxin, reside

on the target membrane whilst other components reside in thecontinuous microvascular endothelium theoreticalmembrane of the free cytoplasmic vesicles (vesicle associatedpore models generally predict the presence of a smallmembrane protein-VAMP). Other components, NSF and soluble2pore population (diameter 10 nm; | 18 units /mm ) NSF attachment protein (a-SNAP) represent two soluble cytosolic

and a large pore population (diameter # 50 nm) of proteins that mediate the docking of free vesicles to targetmuch lower numerical density. The above series of membranes.


of albumin species, and therefore demonstrated an of this function will be our ability to addressassociation between the SNARE trafficking complex questions such as: ‘‘Do caveolae in alveolar epi-and a transport role for caveolae in endothelial cells. thelium detach from the plasmalemma?’’ ‘‘What roleSimilarly, they examined the direct involvement of does the SNARE trafficking complex, or othercertain SNARE components in the intracellular vesicular membrane components, fulfil in the move-endothelial trafficking of caveolae by selectively ment of alveolar epithelial caveolae?’’, ‘‘What con-inhibiting the internalisation of cholera toxin subunit tribution do microtubule-dependent versus microtu-B (a putative caveolar transport marker) through the bule-independent mechanisms play in caveolae de-functional disruption of vesicle associated membrane tachment and trafficking in the thin attenuated re-protein-2 (VAMP-2) [44]. Extending these studies gions of the alveolar type I cell?’’ ‘‘What regulatesSchnitzer and co-workers [43] isolated caveolae the polarised trafficking of caveolae versus theirmembranes from pulmonary microvascular endo- recycling to the original plasma membrane?’’. Thesethelium, and revealed that the caveolae membrane are but a few of the questions that need to bepreparations contained several key components of experimentally addressed. However, it should bethe SNARE complex as well as members of the acknowledged that the in-vivo and in-vitro ex-annexin family (II and IV) and heterotrimeric GTP- perimental models exploited in studies of alveolarbinding proteins, which are both believed to in- epithelium are less amenable to investigation thanfluence plasma membrane dynamics. More recently endothelial models. For example, this is exemplifiedthe same workers [45] have co-localised dynamin, a by the difficulty in animal models of achievingmember of a multigene family of large GTPases, to reproducible and quantifiable solute access to thethe neck of endothelial caveolae and shown its luminal alveolar epithelial surface, and by the lack offunctional involvement in severing the caveolae success so far in being able to isolate and cultureinvagination from the plasma membrane to form alveolar type I epithelial cells (see later).transport vesicles.

As mentioned above, the SNARE protein, VAMP- 4.2. In-vivo kinetic considerations and vesicular2, has been functionally localised to caveolar mem- transportbranes of rat lung microvascular endothelium [44].The same paper reported on the level of VAMP-2 Tracer experiments that attempt to spatially local-specific colloidal gold label associated with the ise solute to morphologically defined structures arealveolar type I epithelial cells within intact lung frequently considered in studies addressing the roletissue. It was noted that VAMP-2 expression was that vesicular transport may serve in the permeabilityevident in the ATI cell although the immunostaining of alveolar epithelium. An implicit need in much ofin the alveolar cell was significantly lower than that the research upon alveolar epithelial permeability is,in the capillary endothelial cell, even when differ- however, the required resolution of mechanisticences in caveolae density between the two cell types information with quantitative transport data, and viceare taken into consideration. This data would not versa.exclude caveolae within the ATI cell from being The recognition of an inverse correlation betweendynamic entities able to detach from a plasma solute molecular size and the rate of transport ormembrane location, as the caveolae (or subpopula- absorption across lung epithelium is substantiated fortions thereof) within alveolar type I epithelium (if a range of molecule classes (including peptides andthey utilise the SNARE complex) may have a proteins), and by a number of different laboratoriesreduced requirement for VAMP-2, or functionally (reviewed in [6]). In many studies, however, theutilise other distinct VAMP related molecules. results are not unequivocally derived from per-

The functional characterisation of endothelial meability data for alveolar epithelium alone, or docaveolae has provided a framework of knowledge not involve investigation of solutes across a par-and study design to allow for the rational characteri- ticularly wide range of molecular weight (MW).sation of caveolae function within alveolar epi- Nevertheless, evidence for an inverse molecularthelium. Fundamental to an improved understanding weight dependency in systemic absorption from the


lung is substantial, and the contributions in the were correct then it would represent a generalisationphysiological literature from Lewis Shanker [47], only, and not exclude the possibility of a significantand Taylor and Garr [48] are perhaps the most role for the receptor-mediated transcytotic transportwidely known. of certain individual macromolecules. In the follow-

Theodore et al. [49] reported the transalveolar ing discussion some consideration has been given totransport of sucrose (MW 342 Da), inulin (5 kDa) the question ‘‘Can the kinetic data for pulmonaryand dextran (MW 60–90 kDa) following direct drug absorption be resolved with a vesicle-mediatedadministration (via catheter) to the terminal airways pathway of transport ? ’’of dogs. These workers observed alveolar per- In addition to specific receptor-mediated trans-meability to decrease with increasing solute molecu- cytosis, macromolecules may also be captured withinlar weight, and the kinetics of absorption to be membrane vesicles via a non-specific adsorptiveconsistent with a first-order process. Effros and membrane binding process, or via simple internalisa-Mason [50] compiled data from a number of pub- tion as part of vesicle-mediated fluid uptake. In thelished in-vivo pulmonary permeability investigations case of both adsorptive and fluid-phase vesicularwhich utilised a range of animal species, involved capture, the vectorial shuttling of individual discretevarious hydrophillic solutes of MW up to 500 kDa, caveolae vesicles transcytosing solute from oneand which were performed using different solute plasma membrane surface to the opposing membraneadministration techniques into the lung. Despite would not appear to fulfil the above observedthese experimental variations the analysis by Effros molecular weight dependence in solute permeabilityand Mason clearly demonstrated solute clearance of alveolar epithelium. This can be argued on thefrom the lung to decrease with increasing molecular basis that fluid-phase uptake while able to displayweight. Within the pharmaceutical sciences Taylor concentration-dependent kinetics, would not showand Farr and co-workers [51,2,3] have generated data discrimination between solutes based upon molecularfrom studies aerosolising into rabbit lung polypep- size. Similarly, under the condition that non-specifictides and proteins of pharmaceutical interest includ- adsorptive solute binding to vesicle membrane doesing, oxytocin (MW 1007 Da), insulin (MW 5.7 kDa), not display saturation, then an adsorptive processhuman growth hormone (MW 22 kDa). Their data would likewise display a concentration-dependenceshowed an inverse relationship between MW and the in solute transport but no molecular weight depen-rate of pulmonary absorption, where the latter was dence. Although in the case of the latter process itdetermined not from ‘time to maximal plasma con- could be envisaged if saturable binding conditionscentration’ (Tmax), but more appropriately from prevailed, and where the binding capacity of thepharmacokinetic calculation of apparent absorption caveolae membranes display differential molar bind-rate constants (ka), which were 6.16, 0.678, and 0.12 ing capacities between solutes of low and high

–1h , respectively. Indeed an important feature in the molecular size, that some molecular size discrimina-comparison of pulmonary kinetic data between sol- tion may well be evident. However, under thisutes and between different studies, is that such condition the transport kinetics would approximate tocomparison is based upon permeability coefficients a concentration-independent, zero-order, process.or absorption rate constants such that differences in If the aim were to resolve the kinetics describeddosing rates or solute clearances are accounted for. above with a vesicular mechanism of pulmonary

The simplistic interpretation of the kinetic data transport then an alternative form of vesicle-me-from the above studies would support that the major diated transfer needs to be considered beyond thatmechanism for protein or macromolecule transport involving the vectorial shuttling of discrete mem-across pulmonary epithelium involves a first-order brane compartments from one plasma membranediffusional process. Upon initial consideration, this surface to another. Once again the discussion istype of kinetics may not appear to conform to a considered with respect to adsorptive or fluid-phasemechanism of macromolecule transport via a trans- capture of solute.cytotic pathway mediated by ATI caveolae-like The numerical density of the caveolae-like vesi-vesicles. Clearly, even if this latter interpretation cles in the ATI cell is high, and in the thin attenuated


regions of the cell the luminal and abluminal mem- remains proportional to their relative diffusion co-brane surfaces are closely opposed, separated by efficients.100–300 nm of cytoplasm. Under these circum- It is evident, however, that certain kinetic featuresstances frequent interactions between vesicles may of pulmonary absorption may not readily be ex-be expected (driven potentially through the forces of plained by any vesicular model. In particular, theBrownian Motion [52,53]) leading to transient inter- inhalational volume-induced increases in the rate ofconnections or fusions between adjacent vesicles. pulmonary solute absorption reported in humans, notWithout giving rise to a complete or continuous only for small molecules such as DTPA [64] andtransepithelial channel or pore, which would allow nedocromil [65], but also for insulin [66]. Thesefor convective transfer and be detrimental to alveolar volume-induced changes in solute absorption do notfluid homeostasis, such transient vesicle interconnec- have to be the result of extreme ventilation con-tions or fusions would provide for a series of ditions such that epithelial membrane damage oc-discontinuous fluid pathways allowing the diffusion curs, e.g. in the insulin report [66] the high volumeof solute (Fig. 3). This fluid pathway would confer inspiratory manoeuvres were limited by the lungan approximate first-order process on solute transfer, vital capacity (averaged 4.1 L) and the low volumewould display MW dependence, and be mediated via manoeuvre controlled by the inhaler device at 2.2 L.a vesicular mechanism, albeit one that does not rely However, the exact mechanism underlying this vol-on the vectorial shuttling of individual discrete ume-induced phenomenon is still to be resolved butvesicles from luminal to abluminal plasmalemmal one hypothesis [66] suggests that increased alveolarsurfaces. An additional condition that would need to expansion leads to changes in paracellular per-be satisfied is that the frequency of interaction and meability as a result of epithelial stretching andfusion between vesicles forming the ‘diffusion path- transient disruption of the tight-junctional complexesway’ is not rate-limiting in terms of solute transfer, between alveolar cells. Only at very high lungi.e. the rate of transcellular passage of solutes distending volumes, as used in animal experimenta-

Fig. 3. A speculative scheme showing how a vesicular transport system may operate in the alveolar epithelium which may afford thepulmonary absorption kinetics for macromolecules to display both a molecular weight dependence and concentration-dependence absorptionrate. Frequent interactions between vesicles leading to transient interconnections or fusions between adjacent vesicles could provide for aseries of discontinuous fluid pathways allowing the diffusion of solute. Solute deposited in alveolar airspace is shown as closed black circles.See text for more details.


tion [67–69], does the alveolar epithelium show endothelial cell. If the membrane vesicle system isexpansion-mediated permeability to large proteins. It maintained at steady-state (i.e. area of vesicle mem-must also be reemphasised that a common mechanis- brane detaching from a plasmalemmal surface istic interpretation of the kinetics of pulmonary ab- balanced by the area of vesicle membrane joining)sorption across a broad range of solute physico- then these transit times would also provide a time-chemical characteristics may obscure the underlying frame for the potential frequency of plasma mem-presence of multiple transport pathways, e.g. for brane vesicle detachment, at least in endothelium.insulin the paracellular pathway may predominate, Clearly the above calculations are based on as-whereas a molecular-weight dependence in pulmon- sumptions not addressed by experimental data withinary transport across a higher range of molecular the alveolar epithelium itself, not least that the ATIweight, e.g. FITC-dextrans of 10–150 kDa [70], may vesicles in the in-vivo cell are able to detach frominvolve a vesicular mechanism. Finally, some evi- the luminal plasmalemma. A considerable researchdence exists [71] indicating that caveolae fulfil a role effort is required to provide a clear understanding ofin the rapid transduction of mechanical signals (flow- the role of caveolae in pulmonary macromoleculeinduced) in vascular endothelium. How caveolae transport. This will involve well designed pulmonaryvesicles within alveolar epithelial cells respond to a pharmacokinetic studies as well as combined cellmechanically-induced stretch stimuli remains to be biology and permeability investigations in appro-examined. priate in-vitro alveolar epithelial cell models.

Given the morphometric information on the den-sities of caveolae-like vesicles in the alveolar epi- 4.3. In-situ tracer studies in alveolar airspacethelial type I cell it is interesting to consider somecalculations on the potential capacity of this vesicu- Despite potential problems with the interpretationlar system for fluid capture from the alveolar air- of tracer experiments useful information on putativespace: a single vesicle represented by a sphere of transport pathways can be provided, information thatdiameter 70 nm would occupy a volume of 1.8 3 has allowed for the further refinement of hypotheses

24 3 21310 m or 1.79 3 10 ml. Morphometric data regarding macromolecule transport pathways in al-indicates a density of approximately 150 vesicles per veolar lung.

2m of luminal ATI surface area, or a total of 375 3 The classical studies of Schneeberger and Kar-

1310 vesicles for an alveolar luminal surface area of novsky [56,57] helped define the alveolar epithelium225 m — a perhaps not unrealistic alveolar epithelial as the limiting restrictive membrane in the alveolar–

drug deposition area that could be achieved follow- pulmonary capillary barrier. Using ultrastructuraling drug delivery with advanced inhaler technology. cytochemical techniques these workers studied theIf it is assumed that for any unit time 5% of the permeability of alveolar epithelium to protein tracers

13vesicles (19 3 10 vesicles) have detached from the such as horseradish peroxidase (HRP) (MW 40luminal membranes of ATI epithelium, then the kDa). They showed that within 90 s of an intraven-

2calculated fluid capture for a 25 m area would ous injection of HRP into adult mice, the HRP hadapproximate to 35 ml per unit time. To put this into passed through the pulmonary endothelial intercellu-perspective a reasonable estimate of the steady-state lar junctions into the underlying basement mem-

2alveolar fluid volume for a 25 m area would be 2–5 brane, but was prevented from gaining access to theml [72]. The issue of speculating upon the frequency alveolar space by the tight-junctional complexes ofof vesicle detachment from the ATI luminal mem- the alveolar epithelium. Both the luminal and ab-brane (i.e. quantifying ‘unit time’ in the above luminal endothelial cell vesicles, (morphologicallycalculation) is, more complicated. Nevertheless, of consistent with caveolae) appeared to contain HRPinterest in this context are predictions [52–55] based reaction product. HRP product was also observed toupon combined morphometric and transport data, or be intracellularly located within endothelial vesiclesthose derived from theoretical modelling, that have that appeared as discrete structures in the cytoplasmgenerated average times (ranging from , 10 s to 5 not attached to the plasma membrane. Only rarelymin) for a caveolae vesicle to traverse a capillary were plasma membrane invaginations in the alveolar


type I cell seen to contain HRP, and these were amounts, by the ATII cells. At the earliest timepointusually connected to the abluminal or interstitial of study (10 min after instillation into the lung)surface of the alveolar epithelial cell. No HRP was clusters of cationic ferritin were observed adhered toseen to access the alveolar airspace even by 60 min the ATII plasmalemma with tracer present withinpost-injection. Similar observations were seen for vesicle compartments proximal to the ATII luminalinjected ferritin. By means of intranasally instilled cell membrane. Within 30–60 min post-instillationperoxidase, Schneeberger and Karnovsky attempted the tracer was trafficked to multi-vesicular bodiesto establish the manner of protein absorption from and lamellar bodies of the ATII cell. At 2 h somethe alveolar airspace. Following this route of ad- tracer was observed in the interstitial space below theministration into the respiratory tract, limited access basal membrane of the ATII cell. Although theof HRP to the alveolar membrane was observed, surfaces of the ATI cell had adhered cationic ferritinwith labelled pinocytic vesicles within the ATI cells particles the numerous vesicles present in the ATIonly rarely demonstrated. By 6 h post instillation cell appeared to be largely devoid of tracer. In thesome alveolar type I cells were seen to contain large same body of work, it was observed that while ATIIvacuoles of HRP reaction product, but no HRP was cells were also seen to internalise instilled dextranobserved discharged into the interstial regions of the (70 kDa), the uptake of this probe by the ATI cellalveolar–capillary barrier. was minimal; neither the ATII or the ATI cell

A range of cationic probes of varying pI values appeared to internalise neutral ferritin. Another studyhave been used as probes to decorate membrane examining cationic ferritin uptake and intracellularsurfaces. In lung studies cationic ferritin has been transport by intact alveolar epithelium of the rat [61],one of the most commonly used tracers. Simionescu also concluded that type I alveolar epithelium inter-and Simionescu [58] studied the differential dis- nalised only very limited amounts of tracer, with thetribution of cell surface charge in alveolar epithelium majority that appeared to be transported to theof mouse lung following airway perfusion with interstitial space following tracheal instillation, doingcationic ferritin. Their results showed that while the so via transport across the type II cell.luminal surface of the ATII cells showed heavy In studies examining the uptake of cationiseddecoration with the cationic probe, the luminal ferritin by alveolar type I cells from goat lung, Atwalsurface of the ATI cell displayed only very light et al. [27] made observations that appeared contrarylabelling. This was interpreted as a difference in the to the cationic ferritin reports described above. Atwaldensity of anionic sites on the respective cell mem- and co-workers instilled cationised ferritin into thebranes, with a relative paucity of anionic charge on right lung via a bronchoscope and observed cationicthe ATI cell surface. A not unrelated series of ferritin decorating the surfaces of both the ATI andinvestigations by these and other workers [59] ATII cells, although the temporal pattern of stainingdescribes in-situ studies addressing the surface indicated that the membranes of the non-coatedcharge and chemistry of endothelial plasmalemmal plasmalemmal invaginations or caveolae of the ATIvesicles. In a variety of endothelial vascular beds it cell were preferentially stained, indicating at theseappears that anionic sites of low pK occur at high sites the presence of highly charged anionic domains.a

density over the entire luminal membrane surface, Within 2 min of instillation the cationic ferritin wasbut are characteristically absent over the large ma- found to be ultrastructurally associated with ATIjority of the membranes of the non-coated plas- vesicles, and within 5 min these vesicles weremalemmal vesicles. If the membranes of the ATI heavily decorated with tracer. They reported evi-plasmalemmal invaginations possess a less anionic dence of discharge of tracer on the abluminal ornature this may have implications for electrostatic interstitial surface of the ATI cell indicative ofinteractions of these domains with proteins in the trancystosis. The authors hypothesised that theiralveolar airspace. divergent findings to that of other studies with

In the rat lung instillation tracer studies of Mary cationic ferritin, reflected a change in the cell surfaceWilliams [60], cationic ferritin was observed to be charge of the ATI cell membranes in goat lunginternalised most rapidly, and in the greatest following exposure of the alveolar epithelial surface


to ruminant gases as part of the natural physiological Within 2 h of albumin instillation they showed ancycling of gas from goat rumen to lung. They increased albumin immunoreactivity in both ATI andpostulated that the induced change in surface charge ATII cells, by this time they also observed that bothmay occur as a mechanism to facilitate the transport inhibitors co-instilled into the airways could reduceout of the lung of ruminal fluids and solutes which this albumin staining to background levels.may also enter the goat lung. Intriguingly, Gordon et Nocodazole significantly increased the numericalal. [62] observed a 5-fold increase in the numerical density of the ATI vesicles by approximately 100%density of non-coated vesicles within the ATI cells without effects upon the size of the vesicles (averageof hamster lung following exposure to NO , sug- diameters 70 nm), indicating a disruption in steady-2

gesting a change had occurred in the steady-state state membrane trafficking by this microtubule inhib-regulation of the cell’s membrane trafficking. These itor; similar effects of nocodazole upon vesicleworkers also reported that with NO exposure the density were seen in the pulmonary capillary endo-2

affinity of anionic surface probes, cationic ferritin thelium. While at the immunocytochemical level theand ruthenium red, for the plasmalemmal surface inhibitors appeared to have an effect upon albuminwas found to increase [63], suggesting a modification association with the alveolar epithelial cells, atin cell surface characteristics. neither 2 nor 8 h post albumin instillation did the

By means of antibodies labelled with HRP, Bignon endocytic inhibitors have an effect upon the alveolar131and co-workers [73] were the first to identify under clearance of albumin ( I-labelled albumin), where

physiological conditions the presence of endogenous clearance was determined by sampling of lungserum proteins, mainly albumin and immunoglobulin lavage fluid or of lung tissue homogenate postG (IgG), within the fluid lining the alveolar epithelial experiment. The authors concluded that while theirsurface. Their immunocytochemical investigations in data would not exclude an endocytic route contribut-rat lung showed these proteins to be present also in ing significantly to the removal of trace protein fromthe non-coated vesicles or invaginations of the ATI the alveolar region, this mechanism is probablycell; at the electron-microscopic level they noted an insufficient to clear large quantities of serum proteinabsence of caveolae-like vesicles in the ATII cell. that may enter the alveolar airspace, as may occur inHastings and co-workers [74,75] explored the clear- hydrostatic pulmonary oedema.ance pathway of native proteins following their In combination, the studies described above usingexogenous instillation into the lungs of rabbits. Their HRP, ferritin, dextran and albumin probes wouldtracer studies [74] showed that alveolar macrophages suggest that the plasmalemmal vesicle system in therapidly (within 2 h) internalise both soluble albumin ATI cell does not fulfil a significant role in macro-and colloidal-gold labelled albumin. By 6 h post- molecule trafficking. However, the interpretation ofinstillation they demonstrated the association, or electron microscopic tracer experiments can be im-apparent internalisation, of soluble albumin within precise, especially with the realisation that dynamicthe caveolae-like vesicles of the ATI cell, and within membrane events such as vesicle internalisation andthe vesicle system of the ATII cell. Neither of the trafficking will continue for some time after thealveolar epithelial cell types appeared to internalise initiation of tissue fixation. Further, using this tech-the colloidal gold conjugated albumin probe, high- nique alone to interpret vesicular labelling in termslighting the differing physico-chemical properties of quantitative vesicular transport is not possible.and biological interactions of the soluble and insolu- Patton [6] raised several reasons why the inhibitorble albumin tracers. studies of Hastings et al. should be interpreted with

In 1994 Hastings and co-workers [75] combined caution, including the possibility that vesicle traffick-morphological tracer techniques with the use of ing in the thin squamous ATI cell might occurvesicular transport inhibitors and the examination of independently of the microtubule network, and thealveolar protein clearances [75]. Among their experi- potential lack of the sustained presence, and hencements, they studied the effects of the microtubule effectiveness, of the endocytic inhibitors in thedisrupting agent, nocodazole, and the endosomal alveolar region over an 8 h period. The role ofacidification inhibitor, monensin, upon the alveolar vesicular-mediated trafficking in the transport ofclearance of soluble albumin from rabbit lung. proteins from alveolar airspace to capillary blood


remains to be established but as yet cannot be niques that localised instilled rhGH to caveolae in ratdismissed. Given what is known about caveolae alveolar epithelium, although Patten and co-workerstransport in other tissues and cell types, it would be indicated that no evidence of hGH receptor expres-surprising if caveolae in alveolar epithelial type I sion could be found on the luminal surface of ratcells did not fulfil a trans-alveolar macromolecule lung alveolar epithelium. In Chinese hamster ovarytrafficking function. (CHO) cells bearing recombinant rhGH-receptor, a

component in the internalisation of rhGH has beenshown to be mediated via caveolae [80]. The kinetics

4.4. Caveolae and receptor-mediated transport of rhGH internalisation in the recombinant CHOcells displayed a bi-phasic response which consisted

An established role for caveolae in the transport of of a relatively more rapid initial period of internalisa-potential inhaled therapeutic macromolecules across tion (5–15 min), followed by a slower uptake phase.the alveolar epithelium remains to be determined. The inhibitor for caveolae formation, the sterolHowever, inferences relating to caveolae functioning binding agent fillipin, reduced internalisation duringin receptor-mediated trancytosis may be gained from the slower component of uptake only.the study of caveolae in cell types of non-alveolar The mechanisms by which chemokines penetratelineage. the alveolar barrier to stimulate systemic granulo-

Insulin is perhaps the most studied protein thera- cytes is of interest to both pharmaceutical andpeutic in regard to pulmonary absorption. The insulin biomedical science disciplines. The studies of Mid-receptor has been localised to caveolae in endothelial dleton et al. [81] addressed the sub-cellular fate of[76] and adipocyte [77] cell types, although the gold labelled IL-8 in venular endothelial cells fol-insulin receptor undergoes dynamic transfer between lowing intradermal injection in rabbits. These work-different membrane domains, including clathrin- ers reported that 30 min after administration the IL-8coated pits. Schnitzer and colleagues [40] have gold-conjugate had become bound to the abluminalshown receptor-mediated endothelial transcytosis of endothelial surface and incorporated into omega-insulin to be undertaken, at least in part, by caveolae. shaped plasma membrane invaginations that provedIn the in-vitro study of Roberts and Sandra [76], to be reactive for caveolin. Following internalisationcomparison was made of the relative contribution of the IL-8 was observed to be transcytosed viaa clathrin-coated pit pathway and a non-coated caveolae to the luminal side of the cell into capillaryvesicle (morphologically identifiable as caveolae) blood. Maybe caveolae within the alveolar type I cellpathway in the transcytosis of insulin across cultured can fulfil a similar role. The Duffy antigen, a broadbovine pulmonary artery endothelial cells. Using spectrum scavenging receptor for chemokines, in-semi-quantitative immunocytochemical analyses they cluding IL-8, has been reported to be localised to thereported that both vesicular populations were associ- caveolae within alveolar epithelium [82].ated with insulin, although a greater (approximately70% of the total) amount of the gold-labelled insulinprobe was associated with caveolae structures. How- 5. Caveolae and caveolin in cultured alveolarever, morphometric evaluation determined the sur- epitheliumface density of clathrin-coated pits in their culturedcell type to approximate only 5% of that for Due to the complex nature of the lung architec-caveolae, leading the authors to interpret that, when ture, the alveolar epithelium is not a readily access-normalised for vesicle density, insulin shows a ible absorption surface to study. Therefore the use ofpreferential interaction with clathrin-coated pits. cultures of alveolar epithelium cells as an in-vitro

The lung delivery of recombinant human growth experimental model for the prediction of the extent,hormone (rhGH) has been investigated, with the rate and mechanism of alveolar absorption of phar-studies [78,79] describing a relatively rapid, and maceuticals has gained acceptance amongst inves-dose-dependent [79], systemic absorption for rhGH. tigators [83].In his review in 1996, Patton [6] alluded to un- Consistent with the in-vivo hypothesis of the ATIIpublished work using immunocytochemical tech- cell transdifferentiating into the in-vivo ATI cell


[18], isolated ATII cells in primary culture grown shown to occur as a function of transdifferentiationover a 5–6 day period on a substratum of tissue of the cultured cell from the ATII to an ATI-‘like’culture plastic loose their characteristic ATII pheno- phenotype [16]. This work showed in freshly isolatedtype and acquire with time the morphology, and rat ATII cells and cells grown to 2 days post-seedingexpression of certain biochemical markers, charac- a lack of caveolae-like structures at the electron-teristic of an ATI-‘like’ phenotype [84,85]. When microscopic level, and a lack or low expression ofgrown on semi-permeable membranes these ATI- caveolin-1 protein. As the ATII cells acquired an‘like’ monolayers generate a restrictive paracellular ATI-‘like’ phenotype with continued primary culturepermeability pathway. Such cultures derived most over a number of days, the expression of caveolin-1commonly from rat have been extensively used in increased, with caveolin-1 signal at day 8 post-the pharmaceutical sciences to investigate the alveo- seeding up to 50-fold greater than at day 2. Inlar transport properties of select macromolecules parallel with the increase in caveolin-1 expression,(reviewed in [83] and [86]). plasmalemmal invaginations characteristic of

A study by Matsukawa et al. [87] evaluated the caveolae (determined morphologically and usingflux of radiolabelled albumin across cultured rat caveolin-1 immunolabelling) became evident in theATI-‘like’ monolayers. The transport rate was much ATI-‘like’ cultures between day 6 and 8 post-seed-faster than that predicted by simple passive diffusion ing. In contrast, when the differentiated ATII pheno-alone, and found to be asymmetric. For example the type was maintained with time by culturing the

14apparent permeability of C-bovine serum albumin freshly isolated ATII cells upon a collagen matrix27 21 27was 0.768 ( 3 10 cm sec ) and 0.39 ( 3 10 with an apical interface of air [38], the temporal

21cm sec ) in the apical to basolateral, and basolateral increase in caveolin-1 expression was not observed,to apical directions, respectively. This led the authors with only very faint signals evident even at day 8to speculate that the likely route of transport for post-seeding, and no generation of caveolae. Al-albumin was via a receptor-mediated transcytotic though parallels between in-vitro and in-vivo ATIIpathway. Kim and co-workers [88] identified on the transdifferentiation remain to be fully defined, the inapical membrane of rat ATI-‘like’ cultures the vitro caveolae and caveolin studies described abovepresence of an albumin binding protein which was correspond to observations in intact lung tissueantigenically similar to the albumin receptor, gp60, showing the presence of caveolae in the in-vivo ATIwhich appears to mediate the transcytosis of native cell and an absence in the in-vivo ATII cell [31].unmodified albumin across capillary endothelium Work from the same laboratory (unpublished) has[42]. The same laboratory [89] examined the per- recently identified components of the SNARE com-meability of the ATI-‘like’ cultures to probes of plex to be expressed in the cultured ATI-‘like’fluid-phase vesicular transport. They found the per- monolayers and for albumin endocytosis by thesemeability coefficients for HRP in both the A → B cells to be modulated by the caveolae inhibitor,and B → A directions to be symmetrical, with per- filipin, without effects upon the internalisation of

29meability coefficients calculated at | 7.0 ( 3 10 transferrin (a probe for clathrin-mediated internalisa-21cm sec ). At 48C the transport of HRP was tion).

decreased by 70% suggesting it’s translocation ac- The ability to isolate primary ATII cells and toross the alveolar cell model did not take place via a culture them to form a polarised monolayer whichparacellular route, but rather by a vesicle-mediated acquires the characteristics of the in vivo ATI cellpathway. has allowed various research groups to study putative

In the report of Cheek et al. [90] the appearance alveolar vectorial electrolyte and drug transportwas noted of a vesicle-like structure attached to the processes. With further characterisation these ATI-plasma membrane of rat ATI-‘like’ monolayer. How- ‘like’ monolayers should provide a suitable in-vitroever, no further characterisation was undertaken. model system to examine the potential traffickingRecently the expression of caveolin-1 and caveolae mechanisms regulating the pulmonary absorption ofbiogenesis within in-vitro rat primary alveolar epi- therapeutic macromolecules, mechanisms that to datethelial monolayer cultures has been reported, and are poorly understood.


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