Plasma Membrane Internalization and Recycling
in Rabbit Lacrimal Acinar Cells
Robert W. Lambert ,*f Carol A. Maves,*-\J. Peter Gieroiu*-\ Richard L. Wood,X and Austin K. Mircheff*\
Purpose. The purpose of this study was to examine internalization and recycling of plasma
membrane constituents in lacrimal gland acinar cells.
Methods. Acinar cells were isolated from rabbit lacrimal glands. Surface-expressed reactive
groups were biotinylated at 4°C with sulfo-N-hydroxysuccinimidyl-biotin. Incorporated biotin
was then labeled with avidin-horseradish peroxidase complex for light microscopy, with avidinlucifer yellow conjugate for fluorescence microscopy, and quantitative fluorometry, and with
avidin-ferritin conjugate for electron microscopy.
Results. At 4°C labels remained at the surfaces of intact cells. Surface avidin-lucifer yellow
decreased markedly, giving way to punctate cytoplasmic labeling, on warming to 37°C. Electron microscopy of cells warmed after labeling with avidin-ferritin revealed ferritin in smooth
vesicles underlying the plasma membranes, in vesicles adjacent to Golgi membranes, and in
multivesicular bodies. Incubation at 37°C before chilling and labeling with avidin-lucifer yellow decreased the cells' capacity to bind avidin-lucifer yellow by 95%, with t 05 < 0.5 min. If
cells were then incubated with avidin-lucifer yellow at 37°C, they took up the marker with a
time course that indicated that 60% of the initial biotin either recycled back to the plasma
membrane or remained in intracellular compartments that could be reached by endocytosed
extracellular fluid. Internalized biotin communicated with extracellular avidin-lucifer yellow
with a t 05 of 2 min, and this process was accelerated by carbachol at concentrations of 10
^mol/1 and 1 mmol/1.
Conclusions. Acinar cell plasma membrane constituents participate in an ongoing, secretagogue-modulated recycling traffic between small surface-expressed pools and 10- to 20-fold
larger intracellular pools. Invest Ophthalmol Vis Sci. 1993;34:305-316.
From the * Departments of Physiology and Biophysics, ^Ophthalmology,
and \Anatomy and Cell Biology, University of Southern California
School of Medicine, Los Angeles, California.
Supported by NIH Grant EY 05801 (AKM), American Heart
Foundation Investigative Group Award 923-IG1, and grants from
the Wright Foundation (RLW), Los Angeles, California, and the
Sjiigren 's Syndrome Foundation (JPG), Port Washington, Neiu York.
Submitted for publication July 22, 1992; accepted September 25, 1992.
Proprietary interest category: N.
Reprint requests: Austin K. Mircheff, Department of Physiology ami
Biophysics, University of Southern California School of Medicine, 1333
San Pablo Street, Los Angeles, CA 90033.
neurotransmitter receptors8'9 are expressed in at least
three different types of membrane population. The
group of membrane populations that most likely represented the basolateral plasma membrane accounted
for only 20% of the membrane-associated alkaline
phosphatase and 10% of the Na,K-ATPase of resting
cells.4 Several populations were major loci of the
trans-Golgi enzyme, galactosyltransferase, suggesting
that they represented a significant Golgi-associated
pool of plasma membrane constituents.15 A third
group of membrane populations, accounting for most
of the remaining Na,K-ATPase, receptor, transporter,
and alkaline phosphatase activities, also contained
Investigative Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2
Copyright © Association for Research in Vision and Ophthalmology
305
JVecent subcellular fractionation analyses of lacrimal
gland fragments and isolated lacrimal acinar cells have
indicated that ion transporters1"7 and secretomotor
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306
Investigative Ophthalmology 8c Visual Science, February 1993, Vol. 34, No. 2
varying amounts of galactosyltransferase. The colocalization of plasma membrane and Golgi markers in
these populations suggested that they might have been
derived from domains of the Golgi complex or other
intracellular structures involved in assembly or recycling of basolateral plasma membrane constituents.4"9
Because newly synthesized Na,K-ATPase subunits
can reach the plasma membrane within 60 to 120
min,10 whereas half-times for Na,K-ATPase turnover
appear to range between 30 and 60 hr,11"14 an intracellular pool containing nearly 90% of the cell's total
Na,K-ATPase would seem too large to consist entirely
of newly synthesized proteins en route to the plasma
membrane. On the other hand, several studies have
raised the possibility that there may be a rapid recycling traffic of acinar cell membrane constituents between intracellular compartments and the basolateral
membranes. Subcellular fractionation analyses of resting and cholinergically stimulated lacrimal gland fragments34 and isolated acini7 suggested that enough
Na,K-ATPase can be translocated from intracellular
pools to the putative basolateral plasma membrane
population to increase the number of plasma membrane-expressed pumps by 30% within 5 min and by
40% within 30 min. Morphologic studies showed that,
in addition to the well known endocytic pathway associated with the retrieval and recycling of secretory vesicle membrane constituents,1516 lacrimal acinar cells
also possess an endocytic pathway that mediates uptake of fluid phase markers from the circulation and so
must originate from the basolateral surface.1718 Recent quantitative analyses of protein release and fluid
phase uptake by acinar cells in suspension have suggested that cholinergic stimulation accelerates the
traffic along both pathways.1920
Taken together, these observations imply a model
in which there is an ongoing recycling traffic of
Na,K-ATPase and other basolateral membrane constituents between small surface-expressed pools and large
intracellular pools. According to this model, cholinergic stimulation induces a remodeling of the basolateral
membrane that is accomplished concomitantly with an
acceleration of the membrane recycling traffic.
Other workers21"24 have used the commercially
available reagent, sulfo-N-hydroxysuccinimidyl-biotin
(sulfo-NHS-biotin), which is water soluble and forms
covalent bonds with superficial free amino groups, to
label proteins expressed in the plasma membranes of
cultured epithelial cells grown on solid substrates and
microporous filters. We have now taken advantage of
this reagent's effective surface specificity and its ability
to form high-affinity complexes with avidin conjugates
to characterize the internalization and recycling of
plasma membrane constituents in suspensions of
freshly isolated lacrimal acinar cells.
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METHODS
Materials and Suppliers
This investigation adhered to the ARVO Statement for
the Use of Animals in Ophthalmic and Vision Research. Male New Zealand albino rabbits (2 kg) were
obtained from Irish Farms (Norco, CA).
Ham's F-12 medium was obtained from Irvine Scientific (Irvine, CA). Purified collagenase was obtained
from Gibco (Gaithersburg, MD). Hyaluronidase was
obtained from Worthington Biochemicals (Freehold,
NJ). Ca2+- and Mg2+-free Hank's medium and DNase
(Type I) were obtained from Sigma (St Louis, MO).
Prosil-28 was obtained from PCR, Inc. (Gainesville,
FL). Sulfo-NHS-biotin and avidin were obtained from
Pierce Chemical Co. (Rockford, IL). Avidin-lucifer
yellow conjugate was from Molecular Probes (Eugene,
OR). Avidin-biotin-horseradish peroxidase complex
(ABC Vecta-Stain), aminoethylcarbazole, and avidinferritin conjugate were from Vector Laboratories
(Burlingame, CA). Other reagents were from standard
suppliers.
Isolation of Acinar Cells
Rabbits were anesthetized with an intramuscular injection of ketamine (40 mg/kg) and xylazine (10 mg/
kg), then killed with a lethal intravenous injection of
sodium pentobarbital. Lacrimal glands were excised
and placed in Ham's F-12 medium supplemented with
penicillin (100 U/ml), streptomycin (0.1 mg/ml), Lglutamine (2 mmol/1), /3-hydroxybutyric acid (2 mmol/
1), linoleic acid (0.084 Mg/ml), HEPES (10 mmol/1), soy
bean trypsin inhibitor (0.05 mg/ml), and bovine serum
albumin (BSA, 0.5%). This medium was kept in a 37°C
shaking water bath under an atmosphere of 95% O2,
5% CO2, and its pH was adjusted to 7.6 with NaOH.
Acinar cells were isolated with the modification of the
collagenase, DNase, and hyaluronidase digestion
methods of Hann et al25 and Oliver,26 described elsewhere.27 After isolation, acinar cells were transferred
to 15-ml conical centrifuge tubes (Falcon Plastics,
Becton Dickinson & Co., Lincoln Park, NJ), resuspended in 4°C Hank's medium supplemented with 10
mmol/1 HEPES (pH 7.6) and 5% BSA, and centrifuged at 100 X g. The cells were resuspended in 4°C
Hank's medium containing 10 mmol/1 HEPES but no
BSA and again centrifuged at 100 X g. The cells were
then resuspended in 4°C Hank's medium containing
10 mmol/1 HEPES, diluted to a concentration of approximately 2 X 107 cells/ml, and allowed to equilibrate on ice for 10 to 15 min. The cell isolation procedure yielded approximately 5 X 107 cells/animal.
Biotinylation
In a typical experiment, cells were divided into two
1.5-ml samples and placed in siliconized 25-ml glass
307
Acinar Cell Plasma Membrane Dynamics
Erlenmeyer flasks. Sulfo-NHS-biotin was added to one
of the samples to a final concentration of 0.15 mg/ml
from a freshly prepared, 10-fold concentrated stock
solution. The biotinylation reaction was allowed to
proceed for 30 min at 4°C, then stopped by dilution
and washing with 50-ml aliquots of 4°C Hank's medium. After three washes, the cells were resuspended
in 1.5 ml of 4°C Hank's medium and divided into 100fi\ aliquots. The nonbiotinylated cell sample was
washed and resuspended in parallel as a control.
Reaction With Avidin Conjugates
In initial experiments the localization of cell-associated biotin was surveyed with avidin-biotin-horseradish peroxidase complex and aminoethylcarbazole.
Cells were resuspended in 4°C Hank's medium containing 5% BSA, then sedimented onto polylysinecoated slides at 800 rpm for 5 min in a Cytocentrifuge
(Shandon, Pittsburgh, PA). Slides were allowed to airdry overnight, then washed for 10 min with phosphate-buffered saline (PBS) containing 1% BSA. They
were incubated with 200 /A avidin-biotin-peroxidase
complex for 20 min and again washed with PBS.
Aminoethylcarbazole, which produces a red precipitate in the presence of peroxidase, was added and incubated for 10 min, followed by Mayer's hematoxylin
for 3 min. The slides were rinsed with tap water for 10
min and mounted in glycerol and PBS.
In all subsequent experiments, each 100-jul aliquot of biotinylated cells was diluted to 500 /i\ with
Hank's medium before the avidin conjugate was
added. In one series of experiments, biotinylated cells
were incubated with avidin-lucifer yellow conjugate
(0.1 mg/ml) at 4°C for 30 min or with avidin-ferritin
conjugate (1:50 final dilution) at 4°C for 90 min. Samples that had not been biotinylated provided estimates
of nonspecific uptake of avidin conjugates. Alternatively, biotinylated cells were incubated with avidin-lucifer yellow in the presence of a 50-fold excess of avidin (5 mg/ml) to yield estimates of the amount of avidin-lucifer yellow taken up by nonsaturable processes.
After reaction with avidin conjugates, cells were
washed by three cycles of centrifugation at 100 X g
and resuspension in 4°C Hank's buffer.
In experiments designed to measure the time- and
temperature-dependent internalization of biotinylated groups, biotinylated cells were warmed by addition of 2 ml of 37°C Ham's F-12 medium, and incubated at 37°C for various intervals. The 37°C incubations were terminated by rapid addition of a twofold
excess of 4°C Hank's medium, and the samples were
placed on ice for reaction with avidin-lucifer yellow, as
described above. The premise of this procedure was
that warming would allow biotinylated membrane constituents to redistribute between surface and intracel-
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lular pools and that the amount of avidin-lucifer yellow bound would provide a measure of the number of
biotinylated groups that remained accessible, either at
the surfaces of intact cells or within damaged cells.
In experiments designed to determine whether internalized biotinylated groups remained in compartments from whence they could communicate with the
extracellular medium, biotinylated cells were warmed
and incubated in Hank's medium for 60 min at 37°C.
Avidin-lucifer yellow conjugate was then added to a
final concentration of 0.1 mg/ml, and the incubation
was allowed to continue for up to 60 min in the presence and absence of 5 mg/ml avidin. Incubations were
terminated by chilling and washing with 4°C Hank's
medium. Initial time values were obtained from cell
samples that were chilled and labeled with avidin-lucifer yellow conjugate at 4°C after 60 min incubation
at37°C.
Quantitation of Cell-Associated Lucifer Yellow
After labeling with avidin-lucifer yellow conjugate and
washing, cells were resuspended in 2-ml aliquots of
4°C Hank's medium. The cells were solubilized by addition of Triton-X 100 from a stock solution of 1% to a
final concentration of 0.1%; the mixtures were then
vortexed vigorously for 5 to 10 sec. Fluorescence was
determined in a Perkin-Elmer (Norwalk, CT) LS5B fluorescence spectrometer with excitation and emission
wavelengths of 428 nm and 540 nm. Data are presented as percentages of the total fluorescence intensity from samples which were incubated with avidin-lucifer yellow conjugate at 4°C with no intervening
warming. All values are means ± SEM from triplicate
determinations, and most experiments were repeated
with at least three acinar cell preparations.
Fluorescence Microscopy
Cells were fixed with 4% paraformaldehyde for 1 hr,
then washed twice in 0.1 mol/1 cacodylate buffer, pH
7.6. The cells were subsequently viewed and photographed on Tri-X pan film, ASA 800 (Kodak, Rochester, NY) with an Olympus Vanox photomicroscope
Model AH-BT, equipped with phase and epifluorescence optics (Olympus, Lake Success, NY).
Electron Microscopy
Cells were fixed with 1% glutaraldehyde in 0.1 mol/1
cacodylate buffer, pH 7.6, then washed with cold
buffer. Postfixation was carried out for 30 min in 2%
aqueous osmium tetroxide. After thorough washing
and dehydration through a graded series of ethanol
concentrations, cells were embedded in Epon 812
(Ladd Research Industries, Inc., Burlington, VT).
Thin sections were cut with a Sorvall MT2B ultramicrotome (RMC, Tucson, AZ), placed on nickel grids
Investigative Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2
308
coated with parlodion, stained with uranyl acetate and
lead citrate, and viewed under a JEOL (Peabody, MA)
1200CX transmission electron microscope at 80 kV.
RESULTS
Specificity of Surface Labeling
The conditions for biotinylating exposed reactive
groups with sulfo-NHS-biotin described under Methods were similar to those used by previous investigators,21"24 and preliminary experiments confirmed that
%
these yielded adequate signals. Because total avidin-lucifer yellow uptake by biotinylated cells should represent a combination of specific binding and nonspecific
adsorption, which might be essentially nonsaturable
and substantial in damaged cells in the preparation,
additional preliminary experiments were performed
to identify an avidin-lucifer yellow concentration that
yielded an optimal ratio of specific to nonspecific labeling.
Intact cells in suspensions that had not been biotinylated did not exhibit any surface labeling with avidin-lucifer yellow (not shown), avidin-ferritin (not
f
• •
# #
4|
|i
FIGURE I. Avidin-biotin-horseradish peroxidase complex
staining of isolated lacrimal
acinar cells. (A) Intact cells in
suspensions that had not been
biotinylated did not show surface labeling. (B) Cells that
were biotinylated, washed, and
fixed, all at 4°C, showed intense surface labeling. (Original magnifications: A, XI40;
B, X180).
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Acinar Cell Plasma Membrane Dynamics
shown), or avidin-biotin-horseradish peroxidase complex {Fig. 1A). In contrast, intact cells in suspensions
that had been biotinylated before incubation with avidin conjugates at 4°C revealed intense, uniform surface labeling with avidin-biotin-horseradish peroxidase complex (Fig. IB) and with avidin-lucifer yellow
(Fig. 2A).
Internalization of Biotinylated Groups
When biotinylated cells were warmed to 37°C for as
little as 30 sec after reaction with avidin-lucifer yellow,
then chilled, rinsed, and fixed, the fluorescent label
appeared to have been internalized, with the surface
label appreciably diminished and a pronounced pattern of punctate cytoplasmic and perinuclear staining
apparent (Fig. 2B). The intensity of the surface labeling was further diminished after 30 min at 37°C
(Fig. 2C).
Electron microscopy of cells that had been labeled
with avidin-ferritin conjugate at 4°C, then either fixed
immediately or warmed to 37°C for various intervals
before fixation, confirmed that the label was specifically localized to and evenly distributed over the cell
surface before warming (Fig. 3A). After 1 min at 37°C,
ferritin was present in smooth-surfaced vesicles near
the plasma membrane (Fig. 3B). After 30 min at 37°C,
the label also could be seen in vesicles near the Golgi
apparatus and in uncoated vesicles and multivesicular
bodies in both the peripheral and the juxtanuclear cytoplasm (Fig. 3C).
A series of experiments were designed to yield
quantitative information about the rate at which biotinylated membrane constituents were internalizated
309
from the cell surface and about their steady-state distribution between surface and intracellular pools.
Cells were warmed and incubated at 37°C for various
intervals before chilling and incubation with avidin-lucifer yellow conjugate at 4°C. In nine separate experiments, the total fluorescence signal decreased by 90%
± 2% (mean ± SEM) after 30 min incubation at 37°C,
The time courses measured in three separate preparations, depicted in Figure 4, indicated that this process
was quite rapid, occurring with a t0 5 of less than 30
sec. In contrast, there was no significant change in the
fluorescence signals from cells that were kept at 4°C
before reaction with avidin-lucifer yellow, then incubated at 37°C for up to 90 min after reaction with the
avidin conjugate (not shown). This result confirmed
that biotinylated groups were not sloughed from the
cells during the course of the experiment. Parallel
measurements with samples that had not been biotinylated yielded a value of 5% ± 2% (n = 4) of the total
initial fluorescence signal obtained from biotinylated
samples.
With allowance for a background signal of 5%, the
difference between the fluorescence signals from cells
which were reacted with avidin-lucifer yellow before
and after 30 min incubation at 37°C would suggest
that 95% of the total initial surface biotin was internalized and that only 5% of the total was still expressed at
the cell surface after 30 min of incubation at 37°C.
Communication of Internalized Biotin With
Extracellular Avidin-Lucifer Yellow
One possible interpretation of the rapid temperaturedependent internalization of biotinylated surface pro-
KiCURE 2. Avidin-lucifer yellow labeling of biotinylaied acinar cells. Cells were biotinylated,
washed, and reacted with avidin-lucifer yellow, all at 4°C, then warmed and incubated at
37°C for various periods, (A) Before wanning, avidin-lucifer yellow intensely stained the
surfaces of intact cells; occasional damaged cells (*) took up the label and exhibited a uniformly bright fluorescence. (B) Cells that had been incubated at 37°C for 30 sec showed
marked diminution of the surface label and a punctate cyLoplasmic and perinuclear pattern
of staining (C) Surface labeling was further diminished in cells that had been incubated at
37°C for 30 min. (Original magnifications: A, X545; B, X510; C, X495.)
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Investigative Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2
310
•vfi
f>
.
;••>,
i
FIGURE 3. Transmission electron micrographs of biotinylated acinar cells. Cells were reacted
with avidin-ferritin conjugate at 4°C, then warmed and incubated at 37°C for various periods. (A) Cells that were fixed without having been warmed showed labeling at the cell
surface (arrowheads). (B) Cells that were incubated at 37°C for 1 min exhibited some surface
labeling but also showed label in smooth surfaced vesicles and multivesicular bodies near the
plasma membrane (arrowheads). (C) Cells incubated at 37°C for 30 min showed label in
vesicles adjacent to the nucleus (N) and in the vicinity of the Golgi apparatus (G). (Original
magnifications: A, X56,250; B, X74,825; C, X90.000.)
teins is that it reflected an ongoing recycling traffic
between the plasma membrane and intracellular compartments. Another possibility was that biotinylated
membrane constituents were somehow recognized as
damaged and, therefore, selectively internalized to be
degraded. A third possibility was that the rapid transition from the biotinylating temperature of 4°C to the
incubation temperature of 37°C induced a burst of
membrane traffic.
An obvious strategy for determining how much of
the internalized biotin could return to the cell surface
would have been to subject the cells to an extended
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series of cycles of incubation at 37°C, chilling, and
reaction with avidin-lucifer yellow at 4°C. Preliminary
experiments, however, indicated that repeated chilling
and rewarming progressively increased the numbers
of damaged cells. Therefore, an alternative strategy,
described under Methods, was designed with the more
modest goal of surveying the ability of internalized
biotin to communicate with avidin-lucifer yellow
added to the extracellular medium. If cells were incubated at 37°C for 60 min to allow biotinylated constituents to be internalized and distributed through the
membrane trafficking pathway, the amount of avidin-
Acinar Cell Plasma Membrane Dynamics
0
2
4
6
8
10
12
14
16
18
20
311
22
24
26
28 30
Time (min)
FIGURE 4. Time course of change in avidin-lucifer yellow
binding after biotinylated (squares) and nonbiotinylated
(crosses) cells were warmed to 37°C. Values are expressed as
percentages of the total fluorescence from biotinylated cells
that were reacted with avidin-lucifer yellow before warming
to 37°C. Each point is the mean ± SEM of triplicate determinations from three separate preparations.
lucifer yellow taken up during a subsequent incubation at 37°C should have represented the sum of four
components: (1) binding to biotinylated groups which
returned to the cell surface; (2) internalization by fluid
phase endocytosis; (3) binding of endocytosed avidinlucifer yellow to biotinylated groups remaining in intracellular compartments; and (4) nonspecific adsorption. In principle, components (1) and (3) should be
saturable with respect to avidin-lucifer yellow concentration, and components (2) and (4) should be nonsaturable.
In a preliminary experiment, depicted in Figure 5,
cells were biotinylated at 4°C, then, still at 4°C,
reacted with 0.1 mg/ml avidin-lucifer yellow in the
presence of increasing concentrations of avidin. Avidin clearly competed with avidin-lucifer yellow for
available binding sites. In three separate experiments,
however, 8% ± 3% of the initial biotin-dependent fluorescence signal was still observed after reaction in the
presence of a 50-fold excess of avidin. This result suggested that the biotinylation procedure may have damaged a small number of cells, so that they took up
avidin-lucifer yellow conjugate by permeation and
nonspecific adsorption as well as by biotin-dependent
binding.
Figure 6A presents time courses of avidin-lucifer
yellow uptake at 37°C by biotinylated cells that had
been pre-equilibrated at 37°C for 60 min. The cells
rapidly took up avidin-lucifer yellow in the absence of
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excess avidin, and this process was markedly suppressed by excess avidin. In contrast, excess avidin had
no significant effect on the initial amount of avidin-lucifer yellow taken up. This observation suggested that
a negligibly small fraction of the biotinylated groups
initially present remained at the cell surface after 60
min of incubation at 37°C. The time course of saturable (ie, avidin-displacable) avidin-lucifer yellow uptake
(Fig. 6B) indicated that a component of the internalized biotin equilibrated with extracellular avidin-lucifer yellow with a t0 5 of approximately 2 min. In seven
separate preparations, the saturable component of
avidin-lucifer yellow uptake after 30 min indicated
that 59% ± 7% of the internalized biotinylated groups
either recycled to the cell surface or remained in intracellular compartments that were accessible to endocytosed fluid. Conversely, 40% of the internalized biotin
must have been sequestered from the endocytic and
recycling pathways.
The hypothesis that a rapid burst of membrane
traffic was triggered by the abrupt transition from 4°C
to 37°C in the experiments summarized in Figure 4
seemed plausible in the light of results from ongoing
studies of fluid phase marker internalization. Fluid
phase uptake is increased twofold if cells are chilled to
4°C immediately before incubation in 37°C media
containing the fluid phase marker. The effect of chilling is transient, because it is reversed within 15 min of
incubation at 37°C (Gierow and Mircheff, unpub-
0
1
2
3
4
Avidin (mg/mg)
FIGURE 5. Competition between avidin and avidin-lucifer
yellow for binding sites on biotinylated (squares) and nonbiotinylated cells (crosses). Each point is the mean ± SEM of
triplicate determinations. In three separate experiments, 8%
± 3% of the biotin-dependent fluorescence signal remained
in the presence of 5 mg/ml avidin.
Investigative Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2
312
2
4
6
8
10
12
14
16
18 20 22
24 26 28 30
60
Time (min)
lished). In the experiments depicted in Figure 6C, cells
that had been biotinylated at 4°C and equilibrated for
60 min at 37°C were cooled back to 4°C for 10 min,
then diluted into 37°C medium containing avidin-lucifer yellow. In four separate experiments, reincubation at 4°C had no significant effect on the subsequent
time course of avidin-lucifer yellow uptake at 37°C.
Thus, the abrupt transition from 4°C to 37°C apparently increases fluid phase endocytosis by some mechanism other than by increasing the amount of membrane recycling per unit time (eg, by altering the surface area:volume relationship of the endocytic
vesicles).
Acceleration of Membrane Traffic by
Carbachol
B
10
12
14
16
18 20 22
24 26 28 30
60
Time (min)
The time- and temperature-dependent communication between internalized biotin and extracellular avidin-lucifer yellow provided an assay for testing the hypothesis that cholinergic stimulation accelerates the
recycling traffic of plasma membrane constituents. As
depicted in Figure 7, stimulation with carbachol markedly accelerated the temperature-dependent uptake
of avidin-lucifer yellow. In three separate experiments, equilibration was 28% ± 5% complete by 0.5
min in the absence of carbachol, 62% ± 6% complete
by 0.5 min in the presence of 10 jumol/1 carbachol, and
67% ± 8% complete by 0.5 min in the presence of 1
mmol/1 carbachol. Because the time courses of uptake
in the presence of carbachol were not linear throughout the first 0.5 min of incubation, the values measured in these experiments must underestimate the
initial uptake rates, which, therefore, must have been
increased more than 2.5-fold by carbachol.
DISCUSSION
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
Time (min)
60
FIGURE 6. Communication between internalized biotin and
avidin-lucifer yellow added to the extracellular medium.
Cells were biotinylated at 4°C, then incubated at 37°C for
60 min before addition of avidin-lucifer yellow. Zero-time
values were obtained from cells that were chilled and
reacted with avidin-lucifer yellow at 4°C after the 60-min
incubation at 37°C. (A) Fluorescence in the absence
(squares) and presence (crosses) of 50-fold excess avidin. (B)
Saturable (avidin-displacable) component from (A). (C) Saturable component of fluorescence from cells with were
chilled and incubated at 4°C after 60 min incubation at
37°C and immediately before dilution into 37°C medium
containing avidin-lucifer yellow. Each point is the mean
± SEM of triplicate determinations, and similar results were
obtained with six additional acinar cell preparations.
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It has been understood since the work of Herzog and
Farquhar15 and Oliver and Hand16 that exocrine
acinar cells endocytically internalize membrane constituents from their apical surfaces. Because fluid
phase markers taken up from the luminal medium appear in the Golgi cisternae as well as in lysosomes, it
appears that at least some of the internalized material
represents secretory vesicle membrane constituents
that are recycled into newly formed secretory vesicles.
Similar studies have yielded evidence that fluid phase
internalization also occurs at the basolateral membranes of exocrine acinar cells.1718 Marker injected
into the circulation appears first in a distinct membrane system concentrated at the base of the cell, then
in structures identified as secondary lysosomes adjacent to the Golgi apparatus. These observations suggested the existence of separate early endocytic systems receiving fluid internalized from the apical and
313
Acinar Cell Plasma Membrane Dynamics
10
12
14
16
18
20
22
24
26
28 30
18
20
22
24
26
28
30
18
20
22
24
26
28
30
Time (min)
10
12
14
16
Time (min)
0
2
4
6
8
10
12
14
16
Time (min)
FIGURE 7. Carbachol-mediated acceleration of saturable avidin-lucifer yellow uptake. Cells were biotinylated, then incubated at 37°C for 60 min before addition of avidin-lucifer
yellow in the presence and absence of excess avidin. (A) Control. (B) 10 )umol/l carbachol was added to incubation media
5 min before addition of avidin-lucifer yellow. (C) 1 mmol/1
carbachol was added 5 min before addition of avidin-lucifer
yellow. Each point is the mean ± SEM of triplicate determinations, and similar results were obtained with two additional acinar cell preparations.
basolateral surfaces, and the general principle of this
spatial organization has been confirmed in several
types of cultured epithelial cells.2829 Moreover, uptake
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into the exocrine cell basolateral endocytic system is,
like retrieval of secretory vesicle membrane constituents from the apical surface, markedly accelerated by
secretagogues.1718
The availability of reagents that covalently attach
biotinyl groups to cell surface proteins has made it
possible to study the trafficking of membrane constituents, which might, through a series of fusion and segregation events, follow intracellular pathways that diverge from the pathway followed by the fluid entrapped during endocytic membrane internalization.
The physical and chemical properties of sulfo-NHSbiotin would appear to make it ideally suited for such a
study. This is due, in part, to the net negative charge
and, therefore, low membrane permeability conferred
by the sulfo group, and, in part, to the biotin moiety,
which has a high affinity (K<, = 10~15 mol/1) for avidin
and so can be readily detected with avidin conjugates.
The use of biotinylating reagents to label membrane
constituents and ligands binding to membrane-associated receptors in cultured epithelial cells is now well
documented,21"24'30'31 and our results confirm the surface specificity of this procedure. We found it necessary to perform the current studies with a preparation
of freshly isolated acinar cells in which there is no topologic distinction between apical and basolateral surfaces. This was because preparations of intact acini,
which retain an obviously polarized intracellular organization,9 contain extracellular matrix material that
binds both fluid phase and membrane surface labels,
and because methods for maintaining rabbit lacrimal
acinar cells in confluent monolayer cultures are not
yet available.
The results we have obtained demonstrate a rapid,
temperature-dependent internalization of biotinylated acinar cell plasma membrane constituents, and
they provide evidence that a significant component of
this internalization represents an ongoing recycling
traffic whose rate is modulated by the secretagogue,
carbachol. With more than 50% of the biotinylated
membrane constituents internalized within 30 sec of
warming to 37°C in the absence of carbachol, the
membrane traffic is remarkably rapid. Its t0 5 is similar
to values for receptor-mediated ligand internalization31'32 and much faster than values for bulk membrane internalization, reported in other cell types.33
The acinar cell membrane recycling pathway must
proceed to and from one or more endocytic compartments without reaching either the Golgi stacks or the
secretory vesicles, neither of which becomes significantly labeled over the course of the experiments.
There do not appear to be substantial populations of
coated pits at the surfaces of acinar cells in situ or in
isolated acini or acinar cell preparations, nor have we
obtained images of surface label in such structures.
314
Investigative Ophthalmology & Visual Science, February 1993, Vol. 34, No. 2
For this reason, it appears that most of the lacrimal
acinar cell membrane constituents entering the endocytic system do so through the alternative endocytic
mechanism described by Sandvig et al,34 West et al,35
and Hansen et al.36
One also can infer that the isolated acinar cells
contain two functionally distinct endocytic compartments, one in which biotinylated constituents continue to communicate, through the ongoing membrane traffic, with the extracellular medium, and one
in which biotinyl groups are sequestered from the
fluid phase. The latter compartment might, conceivably, have contained membrane constituents that were
so altered by the biotinylation procedure that they
were selectively removed from the normal intracellular traffic. Studies of fluid phase uptake by lacrimal
acinar cells have provided evidence for two sequentially loaded endocytic compartments. Access to both
the early and the late compartment is markedly accelerated by carbachol at the optimal concentration of 10
jumol/1, whereas further increasing the carbachol concentration to 1 mmol/1 impairs access to the late compartment.1920 Neither of these compartments is likely
to represent the compartment in which the biotinylated groups are sequestered, because the steady-state
saturable avidin-lucifer yellow uptake is not affected
by carbachol (Fig. 7).
The compartments reached by internalized labeled surface membrane constituents are widely distributed through the cytoplasm (Figs. 3B, C), but they
do not include structures that we would readily identify as lysosomes. In fact, it is possible that many of the
endocytic structures we observe, as well as those previous workers have identified as lysosomes on the basis
of their cytochemically demonstrated acid phosphatase activity,1718 actually represent cross-sections of an
endocytic reticulum such as that noted by Hopkins et
al37 and by Tooze and Hollinshead.38 That such a compartment would contain acid phosphatase, a catalytic
activity typically present in lysosomes, is consistent
with the observation that lysosomal enzymes also are
present in the early endocytic compartments of other
cell types.39 It also may be consistent with our observation that acid phosphatase activity is present in the
group of isolated membrane populations that we have
tentatively identified as originating from intracellular
structures involved in assembly or recycling of basolateral membrane constituents.4"9
The internalization and recycling phenomena that
we have documented would be expected, in principle,
to represent the summation of the traffic along the
two pathways delineated by Oliver18 and Oliver and
Hand.17 Several considerations, however, suggest that
this traffic primarily reflects the movement of membrane constituents that normally are expressed in the
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basolateral plasma membranes. The acinar cell in situ
is a truncated pyramid with a small apex and a large
base. The biotinylation procedure was done with resting cells, that is, cells that had not been stimulated with
carbachol before being chilled to 4°C and biotinylated. Moreover, the appearance of the resting cells at
37°C gives little indication of secretory vesicle exocytosis. Therefore, the composition of the plasma membrane of the isolated cell should have been dominated
by the basolateral membrane constituents.
The membrane constituents that became labeled
by biotin were distributed uniformly over the membrane surface (Fig. 3A), and the observation that 95%
of the total initial biotin was internalized makes it unlikely that this traffic was limited to a set of secretory
vesicle membrane constituents that happened to be
residing in the plasma membrane at the time the cells
were chilled for the biotinylation reaction. Other studies have shown that fluid phase internalization and
protein release are affected differently by increasing
concentrations of carbachol, the former being halfmaximally stimulated at a concentration that has no
significant effect on the latter.20 Finally, in preliminary
subcellular fractionation analyses of cells that had
been biotinylated at 4°C, the label was concentrated in
the membrane populations indicated by previous work
most likely to represent the basolateral membranes
(Maves, Lambert, and Mircheff, unpublished).
Recycling of membrane constituents between a
small pool expressed at the basolateral surface and a
large pool associated with endocytic compartments
certainly is consistent with the picture of acinar cell
organization derived from subcellular fractionation
analyses. It is also consistent with the picture obtained
from fluorescence and electron microscope immunocytochemical studies of the subcellular localization of
Na,K-ATPase subunits.40'41 The stimulation-induced
recruitment of Na,K-ATPase to the basolateral membranes can be seen as resulting from a shift in the
steady-state distribution of Na,K-ATPase between the
two compartments that occurs concomitantly with an
overall acceleration of the recycling traffic. The mechanistic relationship between the acceleration of recycling and the remodeling of the basolateral membrane
is not yet clear. It is possible, however, that the recycling traffic also is important for other aspects of
acinar transport physiology (eg, the internalization
steps in the transcytotic secretion of immunoglobulins, growth factors, such as retinol, and peptide hormones, such as prolactin).42 The acceleration of this
traffic during cholinergic stimulation could account
for the fact that the absolute rate of immunoglobulin
secretion increases markedly with increasing intensity
of stimulation and fluid flow rates.43
As suggested by recent studies with cAMP-me-
Acinar Cell Plasma Membrane Dynamics
dialed endocytosis in hepatocytes, 44 secretagogue control of the basolateral membrane internalization rate
could provide a mechanism for coordinating the transcytotic fluxes of other products with the rate of fluid
and electrolyte secretion.
Finally, the availability of a large intracellular pool
of basolateral membrane constituents might provide
the acinar cell with a mechanism for adjusting its surface area in concert with cell volume oscillations of the
sort that have been documented in other exocrine
acinar cells.45
Key Words
315
13.
14.
15.
16.
aviclin-ferritin, avidin-lucifer yellow, biotin, endocytosis.
References
1. Mircheff AK, Lu CC. A map of membrane populations isolated from rat exorbital gland. Am J Physiol.
1984;247:G651-G661.
2. Mircheff AK, Ingham CE, Lambert RW, Hales KL,
Hensley CB, Yiu SC. Na/H antiporter in lacrimal
acinar cell basal-lateral membranes. Invest Ophthalmol
VisSci. 1987; 28:1726-1729.
3. Lambert RW, Bradley ME, Mircheff AK. Cr/HCO 3 antiporters in rat lacrimal gland. Am J Physiol.
1988;255:G367-G373.
4. Yiu SC, Lambert RW, Bradley ME, et al. Stimulationassociated redistribution of Na+,K+-ATPase in rat lacrimal gland. J MembrBiol. 1988; 102:185-194.
5. Mircheff AK, Bradley ME, Yiu SC, Lambert RW.
Rapid stimulation-associated changes in exocrine
Na,K-ATPase subcellular distribution. Current Topics
in Membrane Transport. 1989;34:121-142.
6. Yiu SC, Wood RL, Mircheff AK. Analytic subcellular
fractionation of acini from rat lacrimal gland. Invest
Ophthalmol VisSci. 1990;31:2437-2447.
7. Yiu SC, Lambert RW, Tortoriello PJ, Mircheff AK.
Secretagogue-induced redistributions of Na,K-ATPase in rat lacrimal acini. Invest Ophthalmol Vis Sci.
1991;32:2976-2984.
8. Bradley ME, Peters CL, Lambert RW, Yiu SC, Mircheff AK. Subcellular distribution of muscarinic acetylcholine receptors in rat exorbital lacrimal gland. Invest Ophthalmol Vis Sci. 1990;31:191-200.
9. Bradley ME, Lambert RW, Lambert RW, Lee LM,
Mircheff AK. Isolation and subcellular fractionation
analysis of acini from rabbit lacrimal gland. Invest Ophthalmol VisSci. 1992;33:2951-2965.
10. Mircheff AK, Bowen JW, Yiu SC, McDonough AA.
Synthesis and translocation of Na,K-ATPase a and j8
subunits to the plasma membrane in MDCK cells. AmJ
Physiol. 1992;262:C470-C483.
11. Lamb JF, Ogden P. Internalization of ouabain and
replacement of sodium pumps in the plasma membranes of HeLa cells following block with cardiac glycosides. QJ Exp Physiol. 1982;67:105-119.
12. Tamkun MM, Fambrough DM. The (Na++K+)-ATPase of chick sensory neurons. Studies on biosynthesis
Downloaded From: http://iovs.arvojournals.org/ on 07/31/2017
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
and intracellular transport. J Biol Chem. 1986; 261:
1009-1019.
Wolitzky BA, Fambrough DM. Regulation of the
(Na++K+)-ATPase in cultured chick skeletal muscle:
modulation of expression by the demand for ion transport. J Biol Chem. 1986; 261:9990-9999.
Rayson BM. Rates of synthesis and degradation of
Na++K+-ATPase during chronic ouabain treatment.
AmJ Physiol. 1989;256:C75-C80.
Herzog V, Farquhar MG. Luminal membrane retrieved after exocytosis reaches most Golgi cisternae
in secretory cells. Proc Natl Acad Sci USA. 1977; 74:
5073-5077.
Oliver C, Hand AR. Uptake and fate of luminally administered horseradish peroxidase in resting and isoproterenol-stimulated rat parotid acinar cells. / Cell,
Biol. 1978; 76:207-220.
Oliver C, Hand AR. Membrane retrieval in exocrine
acinar cells. Methods Cell Biol. 1981;23:429-444.
Oliver C. Endocytic pathways at the lateral and basal
surfaces of exocrine acinar cells./ Cell Biol. 1982;95:
154-161.
Gierow JP, Wood RL, Mircheff'AK. Secretagogue effects on fluid phase marker internalization and protein release in isolated lacrimal acinar cells (abstract).
Invest Ophthalmol Vis Sci. 1991;32(suppl):726.
Gierow JP, Mircheff AK. Effects of carbamylcholine
on internalisation and exocytosis of fluid phase
marker in lacrimal acinar cells (abstract). Invest Ophthalmol VisSci. 1992; 33(suppl): 1289.
Le Bivic A, Real FX, Rodriguez-Boulan E. Vectorial
targeting of apical and basolateral plasma membrane
proteins in a human adenocarcinoma epithelial cell
line. Proc Natl Acad Sci USA. 1989;86:9313-9317.
Lisanti MP, Le Bivic A, Sargiacomo M, RodriguezBoulan E. Steady-state distribution and biogenesis of
endogenous Madin-Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized
cell surface delivery. J Cell Biol. 1989; 109:21172127.
Sargiacomo M, Lisanti M, Graeve L, Le Bivic A, Rodriguez-Boulan E. Integral and peripheral protein
composition of the apical and basolateral membrane
domains in MDCK cells./Membr Biol. 1989; 107:277286.
Le Bivic A, Sambuy Y, Mostov K, Rodriguez-Boulan
E. Vectorial targeting of an endogenous apical membrane sialoglycoprotein and uvomorulin in MDCK
cells. J Cell Biol. 1990; 110:1533-1539.
Hann LE, Tatro JB, Sullivan DA. Morphology and
function of lacrimal gland acinar cells in primary culture. Invest Ophthalmol Vis Sci. 1989;30:145-158.
Oliver C. Isolation and maintenance of differentiated
exocrine gland cells in vitro. In Vitro. 1980; 16:297305.
Mircheff AK, Gierow JP, Lambert RW, Lee LM, Akashi RH, H ofman FM. Class II antigen expression by
lacrimal epithelial cells: an updated working hypothesis for antigen presentation by epithelial cells. Invest
Ophthalmol VisSci. 1991;32:2302-2310.
316
Investigative Ophthalmology 8c Visual Science, February 1993, Vol. 34, No. 2
28. Parton RG, Prydz K, Bomsel M, Simons K, Griffiths G.
37. Hopkins CR, Gibson A, Shipman M, Miller K. MoveMeeting of the apical and basolateral endocytic pathment of internalized ligand-receptor complexes along
ways of the Madin-Darby canine kidney cell in late ena continuous endosomal reticulum. Nature. 1990; 346:
dosomes./ Cell Biol. 1989; 109:3259-3272.
335-339.
29. Hughson EJ, Hopkins CR. Endocytic pathways in po38. Tooze J, Hollingshead M. Tubular early endosomal
larized Caco-s cells: identification of an endosomal
networks in At20 and other cells. J Cell Biol. 1991;
compartment accessible from both apical and basolat115:635-653.
eral surfaces. / Cell Biol. 1990; 110:337-348.
39. Ludwig T, Griffiths G, Hoflack B. Distribution of
30. Morris RE, Saelinger CB. Visualization of intracellunewly synthesized lysosomal enzymes in the endocytic
lar trafficking: use of biotinylated ligands in conjuncpathway of normal rat kidney cells. J Cell Biol.
tion with avidin-gold colloids. J Histochem Cytochem.
1991;15:1561-1572.
1984;32:124-128.
40. Wood RL, Mircheff AK. Apical and basal-lateral
31. Ryan RM, Morris RE, Rice WR, Ciraolo G, Whitsett.
Na,K-ATPase in rat exorbital gland. Invest Ophthalmol
Binding and uptake of pulmonary surfactant protein
Vis Sci. 1986;27:1293-1296.
(SP-A) by pulmonary type II epithelial cells. J Histochem Cytochem. 1989; 37:429-440.
41. Azuma KK, Bradley ME, Wood RL, McDonough AA,
32. Tran D, Carpentier J-L, Sawano F, Gorden P, Orci L.
Mircheff AK. Subcellular distribution of Na,K-ATPLigands internalized through coated or noncoated inase in rat exorbital lacrimal gland. Abstract. J Cell Biol.
vaginations follow a common intracellular pathway.
1990;lll:190.
Proc Natl Acad Sci USA. 1987;84:7957-7961.
42. Mircheff AK, Warren DW, Wood RW, Tortoriello PJ,
33. Steinman RM, Mellman IS, Muller WA, Cohn ZA. EnKaswan RL. Prolactin localization, binding, and efdocytosis and the recycling of plasma membrane. J
fects on peroxidase release in rat exorbital lacrimal
Cell Biol. 1983; 96:1-27.
gland. Invest Ophthalmol Vis Sci. 1992;33:641-650.
34. Sandvig K, Olsnes S, Peterson OW, van Deurs B. Acidi43. Fullard RJ, Tucker DL. Changes in human tear profication of the cytosol inhibits endocytosis from
tein levels with progressively increasing stimulus. Incoated pits./ Cell Biol. 1987; 105:679-689.
vest Ophthalmol Vis Sci. 1991; 32:2290-2301.
35. West MA, Bretscher MD, Watts C. Distinct endocytic
44. Hayakawa T, Bruck R, Ng OC, Boyer JL. DBcAMP
pathways in epidermal growth factor-stimulated hustimulates vesicle traffic and HRP excretion in isoman carcinoma A431 cells. J Cell Biol. 1989; 109:
lated perfused rat liver. Am J Physiol. 1990; 259:
2731-2739.
G727-G735.
36. Hansen SH, Sandvig K, van Deurs B. The pre-endoso45. Wong MMY and Foskett JK. Oscillations of cytosolic
mal compartment comprises distinct coated and nonsodium during calcium oscillations in exocrine acinar
coated endocytic vesicle populations. J Cell Biol.
1991:113:731-741.
cells. Science. 1991; 254:1014-1016.
Downloaded From: http://iovs.arvojournals.org/ on 07/31/2017