Download Caveolae as potential macromolecule trafficking

Advanced Drug Delivery Reviews 49 (2001) 281–300
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Caveolae as potential macromolecule trafficking compartments
within alveolar epithelium
Mark Gumbleton*
Pharmaceutical Cell Biology, Welsh School of Pharmacy, Redwood Building, Cardiff University, Cardiff CF10 3 XF, 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 systemic
delivery of macromolecules, such as therapeutic proteins. The existence of a high numerical density of smooth-coated or
non-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 pulmonary
physiology and pharmaceutical science disciplines. These vesicles, or subpopulations thereof, have been shown to
biochemically possess caveolin, a marker protein for caveolae. This review considers the morphometric and biochemical
studies that have progressed the characterisation of the vesicle populations within alveolar type I epithelium. Parallel
research findings from the endothelial literature have been considered to contrast the state of progress of caveolae research in
alveolar epithelium. Speculation is made on a model of caveolae vesicle-mediated transport that may satisfy some of the
pulmonary pharmacokinetic data that has been generated for macromolecule absorption. The putative transport function of
caveolae 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 of
caveolae in macromolecule transport. A potentially significant role for alveolar caveolae in mediating the alveolar airspace to
blood transport of macromolecules cannot be dismissed. Considerable research is required, however, to address this issue in
a quantitative manner. A better understanding of the membrane dynamics of caveolae in alveolar epithelium will help resolve
the function of these vesicular compartments and may lead to the development of more specific drug targeting approaches for
promoting pulmonary drug delivery.  2001 Elsevier Science B.V. All rights reserved.
Keywords: Caveolin; Caveolae; Lung; Alveolar epithelium; Transport; Endocytosis and transcytosis
Contents
1. Introduction ............................................................................................................................................................................
2. Caveolae and the structural role of caveolins .............................................................................................................................
3. Vesicular system in alveolar epithelium.....................................................................................................................................
3.1. Alveolar epithelial–pulmonary capillary barrier..................................................................................................................
3.2. Membrane vesicles in alveolar epithelium ..........................................................................................................................
3.3. Caveolae and caveolin in alveolar epithelium .....................................................................................................................
*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.
PII: S0169-409X( 01 )00142-9
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M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 – 300
4. Transport role for caveolae in alveolar epithelium......................................................................................................................
4.1. Endothelial paradigm .......................................................................................................................................................
4.2. In-vivo kinetic considerations and vesicular transport .........................................................................................................
4.3. In-situ tracer studies in alveolar airspace ............................................................................................................................
4.4. Caveolae and receptor-mediated transport ..........................................................................................................................
5. Caveolae and caveolin in cultured alveolar epithelium ...............................................................................................................
6. Conclusion .............................................................................................................................................................................
References ..................................................................................................................................................................................
1. Introduction
The permeability characteristics of the lung and
recent advances in inhalational aerosol device technology have led to an increasing interest in exploiting the pulmonary route for the systemic delivery of
macromolecule therapeutics, particularly recombinant proteins and polypeptides [1]. The lung deposition and absorption studies of Colthorpe et al. [2,3]
elegantly demonstrated that the extents of systemic
absorption of insulin or growth hormone following
lung administration positively correlate with the
depth of deposition of these administered proteins
within the lung. Anatomical determinants [4] would
also support the view that the lung periphery, and the
alveolar epithelium in particular, is the appropriate
lung surface to target when aiming to systemically
deliver macromolecules. Some of these anatomical
determinants would include: location of the alveolar
surface beyond the clearance mechanisms of the
mucociliary escalator; the large alveolar surface area
potentially available for absorption; the high blood
flow to the alveolar region, and the thin cellular
barrier from airspace to capillary blood presented by
the alveolar epithelial and pulmonary capillary cells.
Further, in the transport of macromolecules across
the pulmonary alveolar epithelial–capillary endothelial barrier, evidence indicates that it is the
alveolar epithelium that possesses a more restrictive
paracellular pathway than that provided by the
capillary endothelium [5]. As a corollary the mechanisms of transport of macromolecules within alveolar
epithelium are the subject of genuine interest [6], and
in particular the nature and extent of any vesicular
trafficking mechanism(s) such as that potentially
provided by caveolae.
2. Caveolae and the structural role of caveolins
At the electron-microscopic level caveolae are
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most frequently observed as ‘‘smooth coated’’ or
‘‘non-coated’’ omega-shaped invaginations (diameter
of 50–100 nm at the widest point) connected to the
plasmalemma or plasma membrane by a neck-like
structure which affords spatial continuity with the
extracellular environment (Fig. 1a). At least in
endothelial cells caveolae-like vesicles may also be
observed as fused lines or clusters of vesicles at the
plasmalemma [7]. The term ‘‘smooth coated’’ or
‘‘non-coated’’ vesicles has long been used to contrast
them with the electron-dense cytoplasmic coat that
can be seen associated with clathrin-coated pits when
viewed under the electron microscope. However, it is
probable that, both within a single cell and between
cell types, that various subpopulations of smoothcoated or non-coated vesicles exist, and not all will
be caveolae as defined by the presence of the marker
protein caveolin (see later).
Caveolae or caveolae-like structures are recognised as prominent morphological features in a
variety of cell types, notably adipocytes, muscle cells
(skeletal, cardiac and smooth), fibroblasts, capillary
endothelium and type I alveolar epithelial cells,
although to varying extents many other cell types
may possess these morphological structures. A
principal component constituting the striated coat of
caveolae, and a critical structural and functional
element of caveolae, is the cytoplasmically orientated integral membrane protein, caveolin [8]. As a
biochemical marker caveolin has provided for an
additional definition for caveolae beyond that morphological identification alone, i.e. caveolae as flattened caveolin-rich membrane microdomains morphologically indistinguishable from the plasmalemma itself (Fig. 1b).
Caveolin comprises a family of proteins the most
studied of which is caveolin-1. Caveolin-1 appears to
be a critical, but not necessarily the sole, determining
factor in caveolae formation in non-muscle cells
[9–12], with the structural unit for the protein within
the plasma membrane in the form of high molecular
M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 – 300
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Fig. 1. (a) Electron micrograph of a ‘flask-shaped’ caveolae invagination (CV) of diameter approximately 80 nm located within the apical
plasmalemma (PL). The invagination lacks an electron dense cytoplasmic coat characteristic of a clathrin coated pit. At the neck of the
caveolae can be seen a membraneous diaphragm (D) restricting the caveolae opening to 20–40 nm. (b) A schematic representation of
putative 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 possibly
completely close and detach to form a discrete intracellular vesicle.
weight caveolin oligomers [13–15]. The relationship
between caveolin-1 expression and caveolae biogenesis appears to require a threshold level of
caveolin expression for the formation of caveolae
[10,16], such that caveolin expression alone does not
necessarily imply the presence of caveolae within a
cell.
3. Vesicular system in alveolar epithelium
3.1. Alveolar epithelial–pulmonary capillary
barrier
The lower respiratory tract consists of the respiratory bronchioles, the alveolar ducts, and the alveoli
themselves which represent the main location for
gaseous exchange. Alveolar epithelium is predominantly comprised of two cell types, the terminally
differentiated squamous alveolar epithelial type I
(ATI) cell which constitutes approximately 93% of
the alveolar epithelial surface area (33% of alveolar
epithelial cells by number) and the surfactant producing cuboidal alveolar epithelial type II (ATII) cell
comprising the remaining 7% by surface area and
67% by epithelial cell number [17]. The total alveolar epithelial surface area within an average adult
human lung is estimated to be 100–120 m 2 , although
potentially not all of this relatively large surface is
likely to be concurrently available for the absorption
of inhaled drug.
The alveolar epithelial–pulmonary capillary bar-
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M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 – 300
rier comprises the alveolar epithelium and pulmonary
capillary endothelium. In parts of the barrier the
basal membranes of the epithelial and endothelial
cells are directly in contact while in other parts they
separated by interstitium (Fig. 2c). Morphometric
reports [17] indicate that the human ATI cell has an
abluminal or interstitial membrane surface area
averaging 5098 m2 , with an average cell thickness of
0.36 m ranging from 2 to 3 m in the perinuclear
region of the cell to approximately 0.2 m in the
peripheral attenuated regions of the cell. Characteristically the ATI cell displays only sparse cellular
organelles with the majority that are present, located
in the cell’s perinuclear region. Some of these
morphometric features of the ATI cell exemplify the
‘favourable’ anatomical determinants that have
driven interest in the alveolar type I epithelium as a
barrier across which to deliver systemically active
proteins and peptides.
In constrast, the cuboidal ATII cell is considerably
smaller than the ATI cell (e.g. basal surface area
averaging 183 m2 and a uniform cell thickness of
about 5 m [17]) and is richly endowed with organelles and microvilli on it’s apical membrane. Current
evidence would support a role for the ATII cell
serving as the sole in-vivo progenitor for, and
differentiating into, the terminally differentiated ATI
cell (reviewed in [18]).
The pulmonary capillary endothelial cell possesses
a very similar thin attenuated squamous morphology
to the ATI cell, although it’s cell surface area is
reported to be up to three to four times smaller, e.g.
luminal surface area averaging 1353 m2 [17].
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3.2. Membrane vesicles in alveolar epithelium
The existence of a high numerical density of
membrane vesicles within the alveolar epithelial type
I cell has long been recognised. Morphometric data
[19–22] obtained in the early 1980s provided important information on the vesicle populations within
the in-vivo ATI cell. However, while the vesicle
populations studied in these early investigations
encompassed to a high extent smooth- or non-coated
vesicle populations whose morphology is consistent
with caveolae structures, ultrastructural appearance
alone may not be sufficient to functionally define
them as caveolae; the latter necessitating at least
structural association of the vesicle membrane with
caveolin protein. Therefore in the following description (below) of these early studies care has been
taken to avoid defining the vesicles explicitly as
caveolae.
The studies of Gil and co-workers [19,20] described the number and distribution of plasmalemmal
vesicles or invaginations within the ATI cell and
pulmonary capillary endothelial cell of rabbit lung.
These workers did not distinguish between different
types of vesicles but described the presence of high
numbers of non-coated vesicles or invaginations
possessing an average diameter of 70 nm, and in
many cases retaining a neck-like connection to the
plasmalemma. The investigators did not count vesicles that appeared as free discrete entities within the
cytoplasm of the cell with no apparent connection to
either plasmalemmal surface.
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 scanning
microscopy (CLSM). The rat lung tissue was immunostained with anti-caveolin-1 antibody and immunocolloidal gold. The colloidal gold
was 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 moving
through to the bottom of the section. Caveolin-1 staining can be seen along the surfaces of the capillary endothelium and alveolar
epithelium. (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-dimensional
reconstruction to provide a background of lung architecture. The nuclei of the cells are shown overlaid in light grey. The caveolin-1
immunostaining is shown as red / green pairs. The image when viewed through red / green stereo glasses shows the 3-dimensional structure of
the original 10-m thick paraffin section with the profile of caveolin-1 stain. (c) and (d) Transmission electron micrographs of resin-embedded
lung tissue (As – alveolar airspace; C – capillary lumen; S – surfactant). (c) Araldite thin section of tissue postfixed in osmium showing the
alveolar–pulmonary capillary barrier in rat lung. Micrograph shows flask-shaped plasmalemmal invaginations or vesicles in both capillary
endothelium (right-hand surface) and alveolar type I epithelium (left-hand surface); (d) LR White thin section (osmium omitted to retain
antigenicity) immunolabelled for caveolin-1. An attenuated region similar to that in (c) shows anti-caveolin-1 colloidal-gold particles
associated with plasmalemmal invaginations in both the alveolar epithelial and capillary endothelial cells. The epithelial surface is identified
by the presence of surfactant (S).
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M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 – 300
vesicles per m2 of luminal or airspace membrane
surface area, and the reproducible presence of a
significantly higher vesicle load (230 vesicles per m2
cell membrane) at the basal or interstitial membrane
surface. They also determined a vesicle density in
the ATI cell of 145 per m3 cell volume and calculated that approximately 70% of the total plasmalemma surface area of the ATI cell was located within
the membranes of the non-coated plasmalemmal
invaginations. Between species the dimensions of the
ATI cell appear similar [17,23], and based upon a
conservative estimate of ATI cell surface area for the
rabbit, the data of Gil et al. [19,20] would equate to
approximately 600,000 plasmalemmal vesicles or
invaginations on the luminal membrane of the ATI
cell, and in excess of 900,000 on the abluminal
membrane surface; statistics close to that calculated
by the original investigator [20]. Morphometric
studies in dogs by DeFouw and colleagues [21,22]
have similarly reported high vesicle densities (vesicle
diameters 48–60 nm) in the ATI cell with the
number of vesicles per m3 cell volume reported to
average 227–291. In the work of DeFouw [21],
however, a significant differential distribution of
vesicles between the luminal and interstitial membrane surfaces in the ATI cell was not observed.
At first consideration the above statistics for the
ATI cell may appear extreme. Nevertheless, high
numbers of non-coated vesicles or invaginations
morphologically conforming to caveolae are a consistent feature within endothelial microvascular cells.
Reports from a number of laboratories [22,24–26]
each analysing different tissue beds, indicates the
numerical density of such vesicles in capillary
endothelial cells to range from 150 to 600 per m3 cell
volume. Indeed in the above lung morphometric
studies of Gil and coworkers [19,20], data for the
rabbit pulmonary capillary endothelial cell was also
presented, with vesicle numbers reported at 131 per
m3 endothelial cell volume, and vesicle loads upon
each membrane of 196 per m2 for the luminal surface
and 181 per m2 for the abluminal surface. For the
smaller endothelial cell the statistics of Gil and
coworkers [19,20] would equate to approximately
200,000 plasmalemmal vesicles or invaginations on
each of the luminal and abluminal pulmonary capillary endothelial cell membranes.
Clathrin-coated membrane pits or invaginations
are generally readily characterised at the morphologi-
cal level. In none of the early lung alveolar morphometric papers described above were micrographs
presented, or data discussed, pertaining to the presence of electron-dense-coated pits within the in-vivo
ATI cell. Atwal and coworkers in their electron
tracer studies [27] in goat lung alluded to the lack of
clathrin-coated pits in alveolar type I epithelium.
Further, in our own experience of the electron-microscopic analysis of rat and human alveolar type I
epithelium we have failed to identify membrane
structures within the ATI cell conforming to the
morphological characteristics of clathrin-coated pits.
However, an isolated immunoelectron microscopy
report in 1989 [28] localised clathrin light and heavy
chains to the smooth coated vesicles seen in the type
I alveolar epithelial cell. These workers did not
report the morphological presence of clathrin coated
pits in these cells but hypothesised that some components that participate in clathrin-coated pits may
also be involved in the formation of other vesicle
types. This work has not been further substantiated.
The alveolar type I epithelium therefore appears to
parallel the endothelial microvasculature in that the
majority (but not exclusively all) of the vesicles
present in the cell are the smaller non-coated or
smooth-coated vesicle populations that morphologically are recognised as caveolae.
3.3. Caveolae and caveolin in alveolar epithelium
With the discovery of caveolin protein, ultrastructural appearance alone is no longer sufficient to
functionally define smooth-coated or non-coated
plasmalemmal vesicles as caveolae.
In 1994 Lisanti et al. [29] described an association
between caveolin protein expression and the lung.
They isolated caveolin rich domains from whole lung
homogenates by the use of sucrose density gradients.
Based upon ultrastructural evidence and theoretical
calculations they concluded that 80% of the caveolin
signal generated by Western blot analysis is contributed by the ATI cell. However, given the range of
cell types that display caveolae in lung periphery
(e.g. capillary endothelial, fibroblast and alveolar
type I cells) coupled with the architectural complexity and structural diversity of the lung tissue, the
calculation is tenuous.
The work of Kasper et al. in 1998 [30] was the
M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 – 300
first full publication to spatially localise caveolin to
alveolar type I epithelium. Using double immunofluorescence with both frozen and paraffin wax
sections these workers reported the localisation of
caveolin-1 in alveolar type I epithelium of rats and
mini-pigs. In these studies the type II alveolar
epithelial cells were found to be devoid of caveolin-1
staining. Following X-ray irradiation of these animals to induce lung injury, with subsequent initiation
of lung fibrogenesis, Kasper and colleagues noted a
dramatic loss of caveolin-1 expression in the alveolar
epithelium but increases in expression of caveolin-1
in the pulmonary capillary endothelium. They proposed that caveolin-1 may serve as an early indicator
of subcellular alteration during the initial stages of
lung fibrosis.
In 1999 Newman et al. [31] undertook an immunocytochemical study for caveolin in the alveolar
epithelial–pulmonary capillary barrier of rat lung. At
the light microscopic level they used a combination
of bright-field and confocal laser scanning microscopy spatially localise in a 3-dimensional manner
caveolin-1 immunomarker to alveolar epithelial and
pulmonary capillary surfaces of lung tissue (Fig. 2a
and b). At the electron microscopic level they
reported observing a greater number of caveolae-like
structures in the capillary endothelium compared to
that seen in the ATI epithelium (Fig. 2c), although
no quantitative morphometric analyses were undertaken. These workers also noted the absence of
caveolae-like structures in the ATII cell. At the
immunoelectron microscopic level, however, specific
low-level labelling of the ATII cell for caveolin-1
was observed. Both the ATI epithelium and pulmonary capillary endothelium were specifically labelled
with anti-caveolin-1 immunogold particles, with
immunogold particle frequency generally greater in
the endothelium than epithelium. In both cell types
plasmalemmal invaginations could be observed decorated with immunogold label, although not all invaginations were labelled in such a manner (Fig. 2d).
This labelling of some, but not all plasmalemmal
vesicles (despite their morphological similarity) was
considered to reflect either a true biochemical heterogeneity in the smooth-coated vesicle populations
within the ATI cell, or an antigen threshold requirement for caveolin-1 coupled with a variability in the
level of this protein (or of epitope access) between
caveolae structures.
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The biochemical confirmation that the plasmalemmal invaginations or vesicles in the in vivo ATI cell
are caveolae has a number of implications. From a
pharmaceutical perspective it provides a biological
framework for studies addressing the mechanistic
role caveolae vesicles may fulfil in the trafficking of
therapeutic macromolecules across the alveolar–
capillary barrier, and indeed the development of
targeting strategies that could exploit caveolae membrane domains for receptor mediated transcytosis
(see article by Jan Schnitzer in this series). From a
more fundamental basis it should provide insights
into the potential regulation of endogenous solute
trafficking and cell signal regulation within the
alveolar region. The work of Newman et al. [31] and
Kasper et al. [30] has provided some initial characterisation of ATI cell plasmalemma vesicles upon
which their transport role can be further studied with
reference to established functions of caveolae in
other cell types.
4. Transport role for caveolae in alveolar
epithelium
While the morphometric data for the ATI cell
vesicle populations is intriguing in terms of their
putative function as endocytic or transcytotic compartments, direct evidence for their role in transport
within alveolar epithelium is at present extremely
limited. Certainly, the functional characterisation of
ATI vesicle populations lags considerably behind the
progress made in determining a transport role for
caveolae in endothelium. However, until comparatively recently the role of endothelial caveolae in
vesicular trafficking events was itself much debated,
due in part to the lack of recognised inhibitors
specific for caveolae mediated pathways, the lack of
ligands specifically targeting caveolae membranes,
and also a paucity in knowledge of the underlying
mechanisms modulating caveolae membrane vesicle
dynamics. A fuller discussion on the controversy
relating to the role of caveolae in endothelial transport is provided in Jan Schnitzer’s article within this
series. However, to constrast the status of caveolae
transport research in alveolar epithelium to progress
that has been made in the endothelial literature, a
brief overview of the latter is provided below.
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4.1. Endothelial paradigm
Ultrastructural studies by Bungaard et al. [32,33]
and Frøkjaer-Jensen et al. [34,35] undertaking ultrathin serial sectioning with reconstruction revealed
endothelial caveolae to be organised as fused lines or
clusters of vesicles continuous with either the luminal or abluminal plasmalemma. They found a paucity
of free discrete vesicles in the cytoplasm, and argued
as a result that caveolae are merely static invaginations of the cell surface and not dynamic structures
capable of mediating endocytosis or transcytosis.
This work raised the level of debate about the
trafficking function of endothelial caveolae, but did
not provide absolute evidence against such a role,
either via the vectorial shuttling of single discrete
vesicles carrying solute from one plasma membrane
surface to another, or with solute transfer mediated
via transient interconnections or fusions occurring
between lines or clusters of adjacent vesicles as
described by Charles Michel [7,36].
During recent years a growing body of experimental data has led to the general consensus that the
endothelial vesicle system can mediate the transendothelial transport of macromolecules. Of particular note in this context is the work from George
Palade’s laboratory [37–39] and that from the laboratory of Jan Schnitzer [40–45].
The use of electron dense tracer probes to localise
macromolecules to plasma membrane vesicles such
as caveolae, and deduce from this a functional
transport role for the vesicles is impaired by a
number of factors. For example, artifacts may be
introduced during processing of the tissue for microscopic analysis, or by failing to account for the
three-dimensional structure of a cell’s tubulo-vesicular system. Work by Predescu and Palade during the
1990s [37–39], however, exploited a combination of
labelled tracer probes, quantitative permeability investigations, and application of vesicular transport
inhibitors to identify that caveolae could serve as the
structural equivalents of both small and large pores
in continuous microvascular endothelium. For intact
continuous microvascular endothelium theoretical
pore models generally predict the presence of a small
pore population (diameter 10 nm; | 18 units / mm 2 )
and a large pore population (diameter # 50 nm) of
much lower numerical density. The above series of
in-situ studies by Palade and coworkers demonstrated the transcytosis of labelled albumin and
orosomucoid (tracers that qualify as large and small
pore probes, respectively) via endothelial caveolae.
They hypothesised that caveolae could act as large
pores when fully opened, and as small pores when
the neck of the plasma membrane invagination is
constricted to less than 10 nm in diameter. Conversely, caveolae could fulfil such structural equivalents
through the presence of functionally distinct caveolae
subpopulations with a cell.
Using both cultured endothelial monolayers and an
in-situ model of rat lung pulmonary endothelial
microvasculature, Schnitzer and co-workers [40–42]
demonstrated a role for caveolae-mediated transport
in the endothelial permeability to albumin. A specific
binding protein (originally known as gp-60 and
renamed albondin) has been identified on endothelial
surfaces, and appears to mediate the transcytosis of
native albumin, while other endothelial surface binding proteins (gp-18 and gp-30) appear to mediate the
endocytosis of modified albumins [42]. Schnitzer and
coworkers [40] used the sterol binding agent, filipin,
to disassemble caveolae invaginations leading to a
reduction in their surface density to less than 15% of
control, but without effect on the structural integrity
of coated pits. Correspondingly, filipin inhibited, in a
concentration-dependent manner up to 60% of the
transendothelial transport of native albumin across
both in-vitro cultured endothelial monolayers and
in-situ rat lung capillaries. Fillipin treatment exhibited no effect upon paracellular transport pathways as indicated by a lack of effect upon inulin
permeability, or indeed upon the transport of a 2 macroglobulin, a substrate internalised via clathrincoated pits. Further, these workers showed [41] Nethylmaleimide (NEM), an inhibitor of NEM-sensitive factor (NSF)1 to reduce the endothelial transport
1
NSF is a key component in a group of proteins collectively
termed the SNARE complex [46] involved in the subcellular
trafficking and membrane fusion of vesicles. Certain components
of the SNARE machinery, namely SNAP-25 and syntaxin, reside
on the target membrane whilst other components reside in the
membrane of the free cytoplasmic vesicles (vesicle associated
membrane protein-VAMP). Other components, NSF and soluble
NSF attachment protein (a-SNAP) represent two soluble cytosolic
proteins that mediate the docking of free vesicles to target
membranes.
M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 – 300
of albumin species, and therefore demonstrated an
association between the SNARE trafficking complex
and a transport role for caveolae in endothelial cells.
Similarly, they examined the direct involvement of
certain SNARE components in the intracellular
endothelial trafficking of caveolae by selectively
inhibiting the internalisation of cholera toxin subunit
B (a putative caveolar transport marker) through the
functional disruption of vesicle associated membrane
protein-2 (VAMP-2) [44]. Extending these studies
Schnitzer and co-workers [43] isolated caveolae
membranes from pulmonary microvascular endothelium, and revealed that the caveolae membrane
preparations contained several key components of
the SNARE complex as well as members of the
annexin family (II and IV) and heterotrimeric GTPbinding proteins, which are both believed to influence plasma membrane dynamics. More recently
the same workers [45] have co-localised dynamin, a
member of a multigene family of large GTPases, to
the neck of endothelial caveolae and shown its
functional involvement in severing the caveolae
invagination from the plasma membrane to form
transport vesicles.
As mentioned above, the SNARE protein, VAMP2, has been functionally localised to caveolar membranes of rat lung microvascular endothelium [44].
The same paper reported on the level of VAMP-2
specific colloidal gold label associated with the
alveolar type I epithelial cells within intact lung
tissue. It was noted that VAMP-2 expression was
evident in the ATI cell although the immunostaining
in the alveolar cell was significantly lower than that
in the capillary endothelial cell, even when differences in caveolae density between the two cell types
are taken into consideration. This data would not
exclude caveolae within the ATI cell from being
dynamic entities able to detach from a plasma
membrane location, as the caveolae (or subpopulations thereof) within alveolar type I epithelium (if
they utilise the SNARE complex) may have a
reduced requirement for VAMP-2, or functionally
utilise other distinct VAMP related molecules.
The functional characterisation of endothelial
caveolae has provided a framework of knowledge
and study design to allow for the rational characterisation of caveolae function within alveolar epithelium. Fundamental to an improved understanding
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of this function will be our ability to address
questions such as: ‘‘Do caveolae in alveolar epithelium detach from the plasmalemma?’’ ‘‘What role
does the SNARE trafficking complex, or other
vesicular membrane components, fulfil in the movement of alveolar epithelial caveolae?’’, ‘‘What contribution do microtubule-dependent versus microtubule-independent mechanisms play in caveolae detachment and trafficking in the thin attenuated regions of the alveolar type I cell?’’ ‘‘What regulates
the polarised trafficking of caveolae versus their
recycling to the original plasma membrane?’’. These
are but a few of the questions that need to be
experimentally addressed. However, it should be
acknowledged that the in-vivo and in-vitro experimental models exploited in studies of alveolar
epithelium are less amenable to investigation than
endothelial models. For example, this is exemplified
by the difficulty in animal models of achieving
reproducible and quantifiable solute access to the
luminal alveolar epithelial surface, and by the lack of
success so far in being able to isolate and culture
alveolar type I epithelial cells (see later).
4.2. In-vivo kinetic considerations and vesicular
transport
Tracer experiments that attempt to spatially localise solute to morphologically defined structures are
frequently considered in studies addressing the role
that vesicular transport may serve in the permeability
of alveolar epithelium. An implicit need in much of
the research upon alveolar epithelial permeability is,
however, the required resolution of mechanistic
information with quantitative transport data, and vice
versa.
The recognition of an inverse correlation between
solute molecular size and the rate of transport or
absorption across lung epithelium is substantiated for
a range of molecule classes (including peptides and
proteins), and by a number of different laboratories
(reviewed in [6]). In many studies, however, the
results are not unequivocally derived from permeability data for alveolar epithelium alone, or do
not involve investigation of solutes across a particularly wide range of molecular weight (MW).
Nevertheless, evidence for an inverse molecular
weight dependency in systemic absorption from the
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M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 – 300
lung is substantial, and the contributions in the
physiological literature from Lewis Shanker [47],
and Taylor and Garr [48] are perhaps the most
widely known.
Theodore et al. [49] reported the transalveolar
transport of sucrose (MW 342 Da), inulin (5 kDa)
and dextran (MW 60–90 kDa) following direct
administration (via catheter) to the terminal airways
of dogs. These workers observed alveolar permeability to decrease with increasing solute molecular weight, and the kinetics of absorption to be
consistent with a first-order process. Effros and
Mason [50] compiled data from a number of published in-vivo pulmonary permeability investigations
which utilised a range of animal species, involved
various hydrophillic solutes of MW up to 500 kDa,
and which were performed using different solute
administration techniques into the lung. Despite
these experimental variations the analysis by Effros
and Mason clearly demonstrated solute clearance
from the lung to decrease with increasing molecular
weight. Within the pharmaceutical sciences Taylor
and Farr and co-workers [51,2,3] have generated data
from studies aerosolising into rabbit lung polypeptides and proteins of pharmaceutical interest including, oxytocin (MW 1007 Da), insulin (MW 5.7 kDa),
human growth hormone (MW 22 kDa). Their data
showed an inverse relationship between MW and the
rate of pulmonary absorption, where the latter was
determined not from ‘time to maximal plasma concentration’ (Tmax), but more appropriately from
pharmacokinetic calculation of apparent absorption
rate constants (ka), which were 6.16, 0.678, and 0.12
h – 1 , respectively. Indeed an important feature in the
comparison of pulmonary kinetic data between solutes and between different studies, is that such
comparison is based upon permeability coefficients
or absorption rate constants such that differences in
dosing rates or solute clearances are accounted for.
The simplistic interpretation of the kinetic data
from the above studies would support that the major
mechanism for protein or macromolecule transport
across pulmonary epithelium involves a first-order
diffusional process. Upon initial consideration, this
type of kinetics may not appear to conform to a
mechanism of macromolecule transport via a transcytotic pathway mediated by ATI caveolae-like
vesicles. Clearly, even if this latter interpretation
were correct then it would represent a generalisation
only, and not exclude the possibility of a significant
role for the receptor-mediated transcytotic transport
of certain individual macromolecules. In the following discussion some consideration has been given to
the question ‘‘Can the kinetic data for pulmonary
drug absorption be resolved with a vesicle-mediated
pathway of transport ? ’’
In addition to specific receptor-mediated transcytosis, macromolecules may also be captured within
membrane vesicles via a non-specific adsorptive
membrane binding process, or via simple internalisation as part of vesicle-mediated fluid uptake. In the
case of both adsorptive and fluid-phase vesicular
capture, the vectorial shuttling of individual discrete
caveolae vesicles transcytosing solute from one
plasma membrane surface to the opposing membrane
would not appear to fulfil the above observed
molecular weight dependence in solute permeability
of alveolar epithelium. This can be argued on the
basis that fluid-phase uptake while able to display
concentration-dependent kinetics, would not show
discrimination between solutes based upon molecular
size. Similarly, under the condition that non-specific
adsorptive solute binding to vesicle membrane does
not display saturation, then an adsorptive process
would likewise display a concentration-dependence
in solute transport but no molecular weight dependence. Although in the case of the latter process it
could be envisaged if saturable binding conditions
prevailed, and where the binding capacity of the
caveolae membranes display differential molar binding capacities between solutes of low and high
molecular size, that some molecular size discrimination may well be evident. However, under this
condition the transport kinetics would approximate to
a concentration-independent, zero-order, process.
If the aim were to resolve the kinetics described
above with a vesicular mechanism of pulmonary
transport then an alternative form of vesicle-mediated transfer needs to be considered beyond that
involving the vectorial shuttling of discrete membrane compartments from one plasma membrane
surface to another. Once again the discussion is
considered with respect to adsorptive or fluid-phase
capture of solute.
The numerical density of the caveolae-like vesicles in the ATI cell is high, and in the thin attenuated
M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 – 300
regions of the cell the luminal and abluminal membrane surfaces are closely opposed, separated by
100–300 nm of cytoplasm. Under these circumstances frequent interactions between vesicles may
be expected (driven potentially through the forces of
Brownian Motion [52,53]) leading to transient interconnections or fusions between adjacent vesicles.
Without giving rise to a complete or continuous
transepithelial channel or pore, which would allow
for convective transfer and be detrimental to alveolar
fluid homeostasis, such transient vesicle interconnections or fusions would provide for a series of
discontinuous fluid pathways allowing the diffusion
of solute (Fig. 3). This fluid pathway would confer
an approximate first-order process on solute transfer,
would display MW dependence, and be mediated via
a vesicular mechanism, albeit one that does not rely
on the vectorial shuttling of individual discrete
vesicles from luminal to abluminal plasmalemmal
surfaces. An additional condition that would need to
be satisfied is that the frequency of interaction and
fusion between vesicles forming the ‘diffusion pathway’ is not rate-limiting in terms of solute transfer,
i.e. the rate of transcellular passage of solutes
291
remains proportional to their relative diffusion coefficients.
It is evident, however, that certain kinetic features
of pulmonary absorption may not readily be explained by any vesicular model. In particular, the
inhalational volume-induced increases in the rate of
pulmonary solute absorption reported in humans, not
only for small molecules such as DTPA [64] and
nedocromil [65], but also for insulin [66]. These
volume-induced changes in solute absorption do not
have to be the result of extreme ventilation conditions such that epithelial membrane damage occurs, e.g. in the insulin report [66] the high volume
inspiratory manoeuvres were limited by the lung
vital capacity (averaged 4.1 L) and the low volume
manoeuvre controlled by the inhaler device at 2.2 L.
However, the exact mechanism underlying this volume-induced phenomenon is still to be resolved but
one hypothesis [66] suggests that increased alveolar
expansion leads to changes in paracellular permeability as a result of epithelial stretching and
transient disruption of the tight-junctional complexes
between alveolar cells. Only at very high lung
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 the
pulmonary absorption kinetics for macromolecules to display both a molecular weight dependence and concentration-dependence absorption
rate. Frequent interactions between vesicles leading to transient interconnections or fusions between adjacent vesicles could provide for a
series 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.
292
M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 – 300
tion [67–69], does the alveolar epithelium show
expansion-mediated permeability to large proteins. It
must also be reemphasised that a common mechanistic interpretation of the kinetics of pulmonary absorption across a broad range of solute physicochemical characteristics may obscure the underlying
presence of multiple transport pathways, e.g. for
insulin the paracellular pathway may predominate,
whereas a molecular-weight dependence in pulmonary transport across a higher range of molecular
weight, e.g. FITC-dextrans of 10–150 kDa [70], may
involve a vesicular mechanism. Finally, some evidence exists [71] indicating that caveolae fulfil a role
in the rapid transduction of mechanical signals (flowinduced) in vascular endothelium. How caveolae
vesicles within alveolar epithelial cells respond to a
mechanically-induced stretch stimuli remains to be
examined.
Given the morphometric information on the densities of caveolae-like vesicles in the alveolar epithelial type I cell it is interesting to consider some
calculations on the potential capacity of this vesicular system for fluid capture from the alveolar airspace: a single vesicle represented by a sphere of
diameter 70 nm would occupy a volume of 1.8 3
10 24 m3 or 1.79 3 10 213 ml. Morphometric data
indicates a density of approximately 150 vesicles per
m2 of luminal ATI surface area, or a total of 375 3
10 13 vesicles for an alveolar luminal surface area of
25 m 2 — a perhaps not unrealistic alveolar epithelial
drug deposition area that could be achieved following drug delivery with advanced inhaler technology.
If it is assumed that for any unit time 5% of the
vesicles (19 3 10 13 vesicles) have detached from the
luminal membranes of ATI epithelium, then the
calculated fluid capture for a 25 m 2 area would
approximate to 35 ml per unit time. To put this into
perspective a reasonable estimate of the steady-state
alveolar fluid volume for a 25 m 2 area would be 2–5
ml [72]. The issue of speculating upon the frequency
of vesicle detachment from the ATI luminal membrane (i.e. quantifying ‘unit time’ in the above
calculation) is, more complicated. Nevertheless, of
interest in this context are predictions [52–55] based
upon combined morphometric and transport data, or
those derived from theoretical modelling, that have
generated average times (ranging from , 10 s to 5
min) for a caveolae vesicle to traverse a capillary
endothelial cell. If the membrane vesicle system is
maintained at steady-state (i.e. area of vesicle membrane detaching from a plasmalemmal surface is
balanced by the area of vesicle membrane joining)
then these transit times would also provide a timeframe for the potential frequency of plasma membrane vesicle detachment, at least in endothelium.
Clearly the above calculations are based on assumptions not addressed by experimental data within
the alveolar epithelium itself, not least that the ATI
vesicles in the in-vivo cell are able to detach from
the luminal plasmalemma. A considerable research
effort is required to provide a clear understanding of
the role of caveolae in pulmonary macromolecule
transport. This will involve well designed pulmonary
pharmacokinetic studies as well as combined cell
biology and permeability investigations in appropriate in-vitro alveolar epithelial cell models.
4.3. In-situ tracer studies in alveolar airspace
Despite potential problems with the interpretation
of tracer experiments useful information on putative
transport pathways can be provided, information that
has allowed for the further refinement of hypotheses
regarding macromolecule transport pathways in alveolar lung.
The classical studies of Schneeberger and Karnovsky [56,57] helped define the alveolar epithelium
as the limiting restrictive membrane in the alveolar–
pulmonary capillary barrier. Using ultrastructural
cytochemical techniques these workers studied the
permeability of alveolar epithelium to protein tracers
such as horseradish peroxidase (HRP) (MW 40
kDa). They showed that within 90 s of an intravenous injection of HRP into adult mice, the HRP had
passed through the pulmonary endothelial intercellular junctions into the underlying basement membrane, but was prevented from gaining access to the
alveolar space by the tight-junctional complexes of
the alveolar epithelium. Both the luminal and abluminal endothelial cell vesicles, (morphologically
consistent with caveolae) appeared to contain HRP
reaction product. HRP product was also observed to
be intracellularly located within endothelial vesicles
that appeared as discrete structures in the cytoplasm
not attached to the plasma membrane. Only rarely
were plasma membrane invaginations in the alveolar
M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 – 300
type I cell seen to contain HRP, and these were
usually connected to the abluminal or interstitial
surface of the alveolar epithelial cell. No HRP was
seen to access the alveolar airspace even by 60 min
post-injection. Similar observations were seen for
injected ferritin. By means of intranasally instilled
peroxidase, Schneeberger and Karnovsky attempted
to establish the manner of protein absorption from
the alveolar airspace. Following this route of administration into the respiratory tract, limited access
of HRP to the alveolar membrane was observed,
with labelled pinocytic vesicles within the ATI cells
only rarely demonstrated. By 6 h post instillation
some alveolar type I cells were seen to contain large
vacuoles of HRP reaction product, but no HRP was
observed discharged into the interstial regions of the
alveolar–capillary barrier.
A range of cationic probes of varying pI values
have been used as probes to decorate membrane
surfaces. In lung studies cationic ferritin has been
one of the most commonly used tracers. Simionescu
and Simionescu [58] studied the differential distribution of cell surface charge in alveolar epithelium
of mouse lung following airway perfusion with
cationic ferritin. Their results showed that while the
luminal surface of the ATII cells showed heavy
decoration with the cationic probe, the luminal
surface of the ATI cell displayed only very light
labelling. This was interpreted as a difference in the
density of anionic sites on the respective cell membranes, with a relative paucity of anionic charge on
the ATI cell surface. A not unrelated series of
investigations by these and other workers [59]
describes in-situ studies addressing the surface
charge and chemistry of endothelial plasmalemmal
vesicles. In a variety of endothelial vascular beds it
appears that anionic sites of low pKa occur at high
density over the entire luminal membrane surface,
but are characteristically absent over the large majority of the membranes of the non-coated plasmalemmal vesicles. If the membranes of the ATI
plasmalemmal invaginations possess a less anionic
nature this may have implications for electrostatic
interactions of these domains with proteins in the
alveolar airspace.
In the rat lung instillation tracer studies of Mary
Williams [60], cationic ferritin was observed to be
internalised most rapidly, and in the greatest
293
amounts, by the ATII cells. At the earliest timepoint
of study (10 min after instillation into the lung)
clusters of cationic ferritin were observed adhered to
the ATII plasmalemma with tracer present within
vesicle compartments proximal to the ATII luminal
cell membrane. Within 30–60 min post-instillation
the tracer was trafficked to multi-vesicular bodies
and lamellar bodies of the ATII cell. At 2 h some
tracer was observed in the interstitial space below the
basal membrane of the ATII cell. Although the
surfaces of the ATI cell had adhered cationic ferritin
particles the numerous vesicles present in the ATI
cell appeared to be largely devoid of tracer. In the
same body of work, it was observed that while ATII
cells were also seen to internalise instilled dextran
(70 kDa), the uptake of this probe by the ATI cell
was minimal; neither the ATII or the ATI cell
appeared to internalise neutral ferritin. Another study
examining cationic ferritin uptake and intracellular
transport by intact alveolar epithelium of the rat [61],
also concluded that type I alveolar epithelium internalised only very limited amounts of tracer, with the
majority that appeared to be transported to the
interstitial space following tracheal instillation, doing
so via transport across the type II cell.
In studies examining the uptake of cationised
ferritin by alveolar type I cells from goat lung, Atwal
et al. [27] made observations that appeared contrary
to the cationic ferritin reports described above. Atwal
and co-workers instilled cationised ferritin into the
right lung via a bronchoscope and observed cationic
ferritin decorating the surfaces of both the ATI and
ATII cells, although the temporal pattern of staining
indicated that the membranes of the non-coated
plasmalemmal invaginations or caveolae of the ATI
cell were preferentially stained, indicating at these
sites the presence of highly charged anionic domains.
Within 2 min of instillation the cationic ferritin was
found to be ultrastructurally associated with ATI
vesicles, and within 5 min these vesicles were
heavily decorated with tracer. They reported evidence of discharge of tracer on the abluminal or
interstitial surface of the ATI cell indicative of
trancystosis. The authors hypothesised that their
divergent findings to that of other studies with
cationic ferritin, reflected a change in the cell surface
charge of the ATI cell membranes in goat lung
following exposure of the alveolar epithelial surface
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M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 – 300
to ruminant gases as part of the natural physiological
cycling of gas from goat rumen to lung. They
postulated that the induced change in surface charge
may occur as a mechanism to facilitate the transport
out of the lung of ruminal fluids and solutes which
may also enter the goat lung. Intriguingly, Gordon et
al. [62] observed a 5-fold increase in the numerical
density of non-coated vesicles within the ATI cells
of hamster lung following exposure to NO 2 , suggesting a change had occurred in the steady-state
regulation of the cell’s membrane trafficking. These
workers also reported that with NO 2 exposure the
affinity of anionic surface probes, cationic ferritin
and ruthenium red, for the plasmalemmal surface
was found to increase [63], suggesting a modification
in cell surface characteristics.
By means of antibodies labelled with HRP, Bignon
and co-workers [73] were the first to identify under
physiological conditions the presence of endogenous
serum proteins, mainly albumin and immunoglobulin
G (IgG), within the fluid lining the alveolar epithelial
surface. Their immunocytochemical investigations in
rat lung showed these proteins to be present also in
the non-coated vesicles or invaginations of the ATI
cell; at the electron-microscopic level they noted an
absence of caveolae-like vesicles in the ATII cell.
Hastings and co-workers [74,75] explored the clearance pathway of native proteins following their
exogenous instillation into the lungs of rabbits. Their
tracer studies [74] showed that alveolar macrophages
rapidly (within 2 h) internalise both soluble albumin
and colloidal-gold labelled albumin. By 6 h postinstillation they demonstrated the association, or
apparent internalisation, of soluble albumin within
the caveolae-like vesicles of the ATI cell, and within
the vesicle system of the ATII cell. Neither of the
alveolar epithelial cell types appeared to internalise
the colloidal gold conjugated albumin probe, highlighting the differing physico-chemical properties
and biological interactions of the soluble and insoluble albumin tracers.
In 1994 Hastings and co-workers [75] combined
morphological tracer techniques with the use of
vesicular transport inhibitors and the examination of
alveolar protein clearances [75]. Among their experiments, they studied the effects of the microtubule
disrupting agent, nocodazole, and the endosomal
acidification inhibitor, monensin, upon the alveolar
clearance of soluble albumin from rabbit lung.
Within 2 h of albumin instillation they showed an
increased albumin immunoreactivity in both ATI and
ATII cells, by this time they also observed that both
inhibitors co-instilled into the airways could reduce
this albumin staining to background levels.
Nocodazole significantly increased the numerical
density of the ATI vesicles by approximately 100%
without effects upon the size of the vesicles (average
diameters 70 nm), indicating a disruption in steadystate membrane trafficking by this microtubule inhibitor; similar effects of nocodazole upon vesicle
density were seen in the pulmonary capillary endothelium. While at the immunocytochemical level the
inhibitors appeared to have an effect upon albumin
association with the alveolar epithelial cells, at
neither 2 nor 8 h post albumin instillation did the
endocytic inhibitors have an effect upon the alveolar
clearance of albumin ( 131 I-labelled albumin), where
clearance was determined by sampling of lung
lavage fluid or of lung tissue homogenate post
experiment. The authors concluded that while their
data would not exclude an endocytic route contributing significantly to the removal of trace protein from
the alveolar region, this mechanism is probably
insufficient to clear large quantities of serum protein
that may enter the alveolar airspace, as may occur in
hydrostatic pulmonary oedema.
In combination, the studies described above using
HRP, ferritin, dextran and albumin probes would
suggest that the plasmalemmal vesicle system in the
ATI cell does not fulfil a significant role in macromolecule trafficking. However, the interpretation of
electron microscopic tracer experiments can be imprecise, especially with the realisation that dynamic
membrane events such as vesicle internalisation and
trafficking will continue for some time after the
initiation of tissue fixation. Further, using this technique alone to interpret vesicular labelling in terms
of quantitative vesicular transport is not possible.
Patton [6] raised several reasons why the inhibitor
studies of Hastings et al. should be interpreted with
caution, including the possibility that vesicle trafficking in the thin squamous ATI cell might occur
independently of the microtubule network, and the
potential lack of the sustained presence, and hence
effectiveness, of the endocytic inhibitors in the
alveolar region over an 8 h period. The role of
vesicular-mediated trafficking in the transport of
proteins from alveolar airspace to capillary blood
M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 – 300
remains to be established but as yet cannot be
dismissed. Given what is known about caveolae
transport in other tissues and cell types, it would be
surprising if caveolae in alveolar epithelial type I
cells did not fulfil a trans-alveolar macromolecule
trafficking function.
4.4. Caveolae and receptor-mediated transport
An established role for caveolae in the transport of
potential inhaled therapeutic macromolecules across
the alveolar epithelium remains to be determined.
However, inferences relating to caveolae functioning
in receptor-mediated trancytosis may be gained from
the study of caveolae in cell types of non-alveolar
lineage.
Insulin is perhaps the most studied protein therapeutic in regard to pulmonary absorption. The insulin
receptor has been localised to caveolae in endothelial
[76] and adipocyte [77] cell types, although the
insulin receptor undergoes dynamic transfer between
different membrane domains, including clathrincoated pits. Schnitzer and colleagues [40] have
shown receptor-mediated endothelial transcytosis of
insulin to be undertaken, at least in part, by caveolae.
In the in-vitro study of Roberts and Sandra [76],
comparison was made of the relative contribution of
a clathrin-coated pit pathway and a non-coated
vesicle (morphologically identifiable as caveolae)
pathway in the transcytosis of insulin across cultured
bovine pulmonary artery endothelial cells. Using
semi-quantitative immunocytochemical analyses they
reported that both vesicular populations were associated with insulin, although a greater (approximately
70% of the total) amount of the gold-labelled insulin
probe was associated with caveolae structures. However, morphometric evaluation determined the surface density of clathrin-coated pits in their cultured
cell type to approximate only 5% of that for
caveolae, leading the authors to interpret that, when
normalised for vesicle density, insulin shows a
preferential interaction with clathrin-coated pits.
The lung delivery of recombinant human growth
hormone (rhGH) has been investigated, with the
studies [78,79] describing a relatively rapid, and
dose-dependent [79], systemic absorption for rhGH.
In his review in 1996, Patton [6] alluded to unpublished work using immunocytochemical tech-
295
niques that localised instilled rhGH to caveolae in rat
alveolar epithelium, although Patten and co-workers
indicated that no evidence of hGH receptor expression could be found on the luminal surface of rat
lung alveolar epithelium. In Chinese hamster ovary
(CHO) cells bearing recombinant rhGH-receptor, a
component in the internalisation of rhGH has been
shown to be mediated via caveolae [80]. The kinetics
of rhGH internalisation in the recombinant CHO
cells displayed a bi-phasic response which consisted
of a relatively more rapid initial period of internalisation (5–15 min), followed by a slower uptake phase.
The inhibitor for caveolae formation, the sterol
binding agent fillipin, reduced internalisation during
the slower component of uptake only.
The mechanisms by which chemokines penetrate
the alveolar barrier to stimulate systemic granulocytes is of interest to both pharmaceutical and
biomedical science disciplines. The studies of Middleton et al. [81] addressed the sub-cellular fate of
gold labelled IL-8 in venular endothelial cells following intradermal injection in rabbits. These workers reported that 30 min after administration the IL-8
gold-conjugate had become bound to the abluminal
endothelial surface and incorporated into omegashaped plasma membrane invaginations that proved
to be reactive for caveolin. Following internalisation
the IL-8 was observed to be transcytosed via
caveolae to the luminal side of the cell into capillary
blood. Maybe caveolae within the alveolar type I cell
can fulfil a similar role. The Duffy antigen, a broad
spectrum scavenging receptor for chemokines, including IL-8, has been reported to be localised to the
caveolae within alveolar epithelium [82].
5. Caveolae and caveolin in cultured alveolar
epithelium
Due to the complex nature of the lung architecture, the alveolar epithelium is not a readily accessible absorption surface to study. Therefore the use of
cultures of alveolar epithelium cells as an in-vitro
experimental model for the prediction of the extent,
rate and mechanism of alveolar absorption of pharmaceuticals has gained acceptance amongst investigators [83].
Consistent with the in-vivo hypothesis of the ATII
cell transdifferentiating into the in-vivo ATI cell
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M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 – 300
[18], isolated ATII cells in primary culture grown
over a 5–6 day period on a substratum of tissue
culture plastic loose their characteristic ATII phenotype and acquire with time the morphology, and
expression of certain biochemical markers, characteristic of an ATI-‘like’ phenotype [84,85]. When
grown on semi-permeable membranes these ATI‘like’ monolayers generate a restrictive paracellular
permeability pathway. Such cultures derived most
commonly from rat have been extensively used in
the pharmaceutical sciences to investigate the alveolar transport properties of select macromolecules
(reviewed in [83] and [86]).
A study by Matsukawa et al. [87] evaluated the
flux of radiolabelled albumin across cultured rat
ATI-‘like’ monolayers. The transport rate was much
faster than that predicted by simple passive diffusion
alone, and found to be asymmetric. For example the
apparent permeability of 14 C-bovine serum albumin
was 0.768 ( 3 10 27 cm sec 21 ) and 0.39 ( 3 10 27
cm sec 21 ) in the apical to basolateral, and basolateral
to apical directions, respectively. This led the authors
to speculate that the likely route of transport for
albumin was via a receptor-mediated transcytotic
pathway. Kim and co-workers [88] identified on the
apical membrane of rat ATI-‘like’ cultures the
presence of an albumin binding protein which was
antigenically similar to the albumin receptor, gp60,
which appears to mediate the transcytosis of native
unmodified albumin across capillary endothelium
[42]. The same laboratory [89] examined the permeability of the ATI-‘like’ cultures to probes of
fluid-phase vesicular transport. They found the permeability coefficients for HRP in both the A → B
and B → A directions to be symmetrical, with permeability coefficients calculated at | 7.0 ( 3 10 29
cm sec 21 ). At 48C the transport of HRP was
decreased by 70% suggesting it’s translocation across the alveolar cell model did not take place via a
paracellular route, but rather by a vesicle-mediated
pathway.
In the report of Cheek et al. [90] the appearance
was noted of a vesicle-like structure attached to the
plasma membrane of rat ATI-‘like’ monolayer. However, no further characterisation was undertaken.
Recently the expression of caveolin-1 and caveolae
biogenesis within in-vitro rat primary alveolar epithelial monolayer cultures has been reported, and
shown to occur as a function of transdifferentiation
of the cultured cell from the ATII to an ATI-‘like’
phenotype [16]. This work showed in freshly isolated
rat ATII cells and cells grown to 2 days post-seeding
a lack of caveolae-like structures at the electronmicroscopic level, and a lack or low expression of
caveolin-1 protein. As the ATII cells acquired an
ATI-‘like’ phenotype with continued primary culture
over a number of days, the expression of caveolin-1
increased, with caveolin-1 signal at day 8 postseeding up to 50-fold greater than at day 2. In
parallel with the increase in caveolin-1 expression,
plasmalemmal invaginations characteristic of
caveolae (determined morphologically and using
caveolin-1 immunolabelling) became evident in the
ATI-‘like’ cultures between day 6 and 8 post-seeding. In contrast, when the differentiated ATII phenotype was maintained with time by culturing the
freshly isolated ATII cells upon a collagen matrix
with an apical interface of air [38], the temporal
increase in caveolin-1 expression was not observed,
with only very faint signals evident even at day 8
post-seeding, and no generation of caveolae. Although parallels between in-vitro and in-vivo ATII
transdifferentiation remain to be fully defined, the in
vitro caveolae and caveolin studies described above
correspond to observations in intact lung tissue
showing the presence of caveolae in the in-vivo ATI
cell and an absence in the in-vivo ATII cell [31].
Work from the same laboratory (unpublished) has
recently identified components of the SNARE complex to be expressed in the cultured ATI-‘like’
monolayers and for albumin endocytosis by these
cells to be modulated by the caveolae inhibitor,
filipin, without effects upon the internalisation of
transferrin (a probe for clathrin-mediated internalisation).
The ability to isolate primary ATII cells and to
culture them to form a polarised monolayer which
acquires the characteristics of the in vivo ATI cell
has allowed various research groups to study putative
alveolar vectorial electrolyte and drug transport
processes. With further characterisation these ATI‘like’ monolayers should provide a suitable in-vitro
model system to examine the potential trafficking
mechanisms regulating the pulmonary absorption of
therapeutic macromolecules, mechanisms that to date
are poorly understood.
M. Gumbleton / Advanced Drug Delivery Reviews 49 (2001) 281 – 300
6. Conclusion
There is a genuine clinical and commercial interest
in exploiting the pulmonary route for the systemic
delivery of macromolecules, particularly recombinant proteins and polypeptides. The alveolar epithelium appears to be the appropriate lung surface to
target when aiming to systemically deliver macromolecules. The existence of a high numerical density
of smooth-coated or non-coated plasma membrane
vesicles or invaginations within the alveolar epithelial type I cell has long been recognised. These
vesicles, or subpopulations thereof, have been shown
to biochemically possess caveolin, a marker protein
for caveolae. From what is known about caveolae
function in other cell types a role within alveolar
type I epithelium for the endocytic and / or transcytotic trafficking of solute by caveolae may be
expected. Pulmonary pharmacokinetic data and
tracer experiments within the alveolar airspace have
tended to support the view that alveolar vesicle
mediated trafficking will in general function as a
minor pathway in protein absorption. A model, not
inconsistent with some of the pulmonary pharmacokinetic data, of how alveolar epithelial vesicles
may serve as a pathway for macromolecule transport
can be envisaged, and a potentially significant role
for alveolar caveolae in mediating the alveolar
airspace to blood transport of macromolecules cannot
be dismissed. A better understanding of the membrane dynamics of caveolae in alveolar epithelium
will help resolve the function of these vesicular
compartments and may lead to the development of
more specific drug targeting approaches for promoting pulmonary drug delivery.
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