1 Department of Basic Medical Sciences, School of Veterinary Medicine, and 2 Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patch clamp experiments were conducted to study the effects of basal lamina (basement membrane) of chicken ovarian follicle on membrane Ca2+ currents in differentiated chicken granulosa cells in a homologous system. The whole cell patch clamp technique was used to simultaneously monitor membrane capacitance (an indirect measure of total cell surface area) and currents flowing through voltage-dependent Ca2+ channels (using Ba2+ as the charge carrier). Membrane capacitance was smaller in cells incubated on intact basal lamina than in control cells (incubated on tissue culture-treated plastic substratum). Granulosa cells expressed both T- and L-type Ca2+ currents, and the amplitudes of the currents in cells incubated on intact basal lamina were significantly lower than those of control cells. Also, granulosa cells incubated on intact basal lamina were found to have significantly lower T- or L-type Ca2+ current densities than control cells. Intact basal lamina that had been stored for 12 mo produced effects on T- and L-type Ca2+ currents similar to those caused by freshly isolated basal lamina. The basal lamina was solubilized completely in one step and used to coat glass coverslips (uncoated glass coverslips served as controls). Granulosa cells incubated on coverslips precoated with solubilized basal lamina assumed spherical shape similar to those incubated on intact basal lamina. Similar to the observations made for intact basal lamina, the solubilized basal lamina suppressed T- and L-type Ca2+ currents in the differentiated granulosa cells. Moreover, fibronectin, laminin, and type IV collagen, obtained from commercial sources, attenuated T- and L-type Ca2+ currents in the differentiated granulosa cells. This interplay between basal lamina and Ca2+ currents may be one mechanism that subserves the effects of the matrix material on metabolic functions of granulosa cells.
basement membrane; extracellular matrix; calcium current; calcium channel; patch clamp; ovary; chicken; fibronectin; laminin; type IV collagen
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
BASEMENT MEMBRANES (basal laminae) are specialized extracellular matrix sheets that provide mechanical support and important signals for growth and differentiation to cells with which they are associated (72). The components of basement membrane obtained from nonovarian sources regulated the morphology and steroidogenesis in granulosa cells from rat (1-3, 9, 28) and human (27) ovaries. Similarly, it was shown recently that basal lamina of the chicken ovarian follicle regulated cell shape and progesterone synthesis in chicken granulosa cells (5, 7). However, the signaling mechanisms involved in the actions of basement membrane (basal lamina) in granulosa cells are not fully known.
The results of studies with porcine (63), rat (22, 61), or chicken (6) granulosa cells showed that basal and gonadotropin-stimulated steroid hormone biosynthesis required the obligatory presence of Ca2+ in the external medium. In addition, external Ca2+ is required for steroid hormone-induced protein synthesis in avian granulosa cells (17). Experiments with radioisotopes demonstrated transmembrane Ca2+ exchange in porcine granulosa cells (62), and studies with Ca2+-sensitive fluorescent dyes revealed Ca2+ influx into rat (66), chicken (32, 49), and porcine (23-25) granulosa cells. Furthermore, experiments conducted with patch clamp techniques showed that avian and mammalian granulosa cells express Ca2+-specific channels (57) and Ca2+ currents (14, 38, 58, 65).
Because basement membrane/basal lamina is an important regulator of progesterone synthesis in granulosa cells (1-3, 5, 9) and because external Ca2+ is required for the regulation of metabolic functions in granulosa cells, it was hypothesized that basal lamina modulates the functions of Ca2+ channels in granulosa cells. To examine the effects of basal lamina on transmembrane Ca2+ transport in granulosa cells in a homologous system, it is necessary to obtain a pure preparation of intact basal lamina from the ovarian follicle. The avian ovarian follicle was used as a model system because its unique anatomic structure permitted the isolation of pure and intact basal lamina and granulosa cells. In the avian ovarian follicle, the granulosa layer (membrana granulosa) consists of a single layer of granulosa cells located between the basal lamina and the perivitelline layer (10, 52, 53), making possible the isolation of intact basal lamina in a hypotonic solution (7). In the present study, the effect of basal lamina (obtained from the largest preovulatory chicken follicle) on differentiated granulosa cells (isolated from the preovulatory follicle) was examined in a homologous system.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chemicals. HEPES, collagenase type IV, soybean trypsin inhibitor, trypsin, BSA (fraction V), penicillin G, streptomycin, fungizone, aspartic acid, N-methyl-D-glucamine (NMDG), adenosine-triphosphate-magnesium (ATP-Mg), tetraethyl ammonium chloride (TEA), nifedipine, EGTA, mouse type IV collagen, mouse laminin, cycloheximide, actinomycin D, inorganic salts, and Trizma base were purchased from Sigma Chemical (St. Louis, MO). Medium 199 (M199), containing Hanks' salts, was from GIBCO-BRL (Grand Island, NY). Flunarizine was obtained from Calbiochem (San Diego, CA). Human cellular fibronectin was obtained from Upstate Biotechnology (Lake Placid, NY).
Animals. Single-Comb White Leghorn hens, obtained from Purdue University Poultry Research Farms (West Lafayette, IN) in their first year of reproductive activity, were caged individually in a windowless, air-conditioned room with a 14:10-h light-dark cycle. They had free access to a layer ration and tap water. The time of egg lay of each bird in the colony was noted daily to the nearest 30 min. The layers were injected with ketamine (50 mg/kg body wt) 10 min before hens were killed by cervical dislocation ~10-12 h before the expected time of ovulation of the largest preovulatory follicle (F1). The F1 was removed and placed in ice-cold Hanks' basic salt solution (HBSS) containing 140 mM NaCl, 5 mM KCl, 1.1 mM MgCl2, 2.5 mM CaCl2, 10 mM HEPES, and 5.6 mM glucose (pH 7.4). The theca and granulosa cell layers (membrana granulosa) were separated by the method of Gilbert et al. (29).
Isolation of intact basal lamina. Basal lamina was isolated as previously described (7). Briefly, the granulosa cell layer obtained from the F1 was placed in a hypotonic solution containing 10 mM Tris · HCl (pH 7.4), 0.5 mg/l leupeptin, 1 mM EDTA-Na2, 0.7 mg/l pepstatin, and 0.2 mM phenylmethylsulfonyl fluoride in a petri dish. The granulosa cells, sandwiched between the basal lamina and perivitelline layer, were lysed hyposmotically, and the basal lamina and perivitelline layer were separated. This basal lamina of avian ovarian follicle (BLAOF) preparation is intact and complete. The side of basal lamina that was in contact with granulosa cells in situ was designated the "granulosa side," and the side in contact with theca tissue was designated the "theca side."
Solubilization of the basal lamina.
Basal laminae were solubilized in one step. They were placed in a
microfuge tube, and solubilization buffer containing 6 M guanidine-HCl,
50 mM Tris · HCl (pH 7.4), and 5% -mercaptoethanol was
added (100 µl per basal lamina). After shaking for 60 min at 4°C,
the entire basal lamina was solubilized (referred to as "total
fraction"). Exclusion of
-mercaptoethanol from the solubilization buffer led to incomplete solubilization of the basal lamina (fragments remained). The solubilized material was placed in a 3-kDa cutoff dialysis membrane and dialyzed against 150 mM NaCl and 50 mM
Tris · HCl (pH 7.4) at 4°C overnight; it turned cloudy
because of precipitation of proteins.
Preparation of intact basal lamina-containing dishes for cell culture. For experiments designed to study the effects of intact basal lamina on membrane currents, pieces of the intact basal lamina (2-3 cm2) were spread in 35-mm tissue culture dishes (Corning, Corning, NY) and allowed to dry in a laminar flow hood. Blank tissue culture-treated plastic substratum (plastic substratum) served as control. Unless stated otherwise, the experiments were conducted with cells incubated on the granulosa side of the intact basal lamina. The intact basal lamina-containing dishes were either used immediately or wrapped in aluminium foil and stored at 4°C. Tissue isolation and preparation of culture dishes were carried out under sterile conditions.
Preparation of solubilized basal lamina-coated coverslips for cell culture. The solubilized basal lamina (total fraction) was diluted with deionized water, and aliquots of 100-200 µl containing 5-50 µg of protein were spread on a 12-mm-round glass coverslip (Warner Instrument, Hamden, CT) and allowed to dry under a tissue culture hood (precoated coverslips). Glass coverslips that received only vehicle served as controls. The precoated coverslips were either used immediately or wrapped in aluminium foil and stored at 4°C. Tissue isolation, solubilization, dialysis, and preparation of coverslips were carried out under sterile conditions.
Preparation of fibronectin-, laminin-, and type IV collagen-coated dishes for cell culture. Fibronectin, laminin, or collagen IV was diluted with deionized water, and aliquots of 1-2 ml containing 5-50 µg of protein were spread in Corning 32-mm culture dishes and allowed to dry under a tissue culture hood (precoated culture dishes). Culture dishes that received only vehicle served as controls.
Granulosa cell culture. Granulosa cells were dispersed in M199 containing 350 mg/l NaHCO3, 10 mM HEPES, collagenase (500,000 U/l), and trypsin inhibitor (200 mg/l) at pH 7.4 (51). Cell viability, determined by the trypan blue exclusion method, was routinely >95%. Granulosa cells were plated in intact basal lamina-containing dishes or in 35-mm dishes containing coverslips that were precoated with the total fraction of solubilized basal lamina. The cells were incubated for 1-24 h at 37°C in serum-free M199 containing 0.1% (wt/vol) BSA, 350 mg/l NaHCO3, and HEPES 10 mM (pH 7.4) (51).
Electrophysiology.
To record Ca2+ currents, the pipette contained 140 mM NMDG,
140 mM aspartate, 5 mM EGTA, 2 mM ATP-Mg, 1 mM MgCl2, and
10 mM HEPES at pH 7.2; the bath solution contained 127 mM NMDG, 127 mM
aspartate, 0.086 mM CaCl2, 1 mM MgCl2, 5 mM
TEA, 10 mM barium acetate, 5.6 mM glucose, and 10 mM HEPES at pH 7.4. The conventional whole cell recording method was used in this study.
Recording pipettes were fabricated from borosilicate filament glass
(Warner Instrument). Electrode resistance was between 3 and 9 M in
the bath solution. Unless noted otherwise, the linear (leak) component of the total membrane current was subtracted by extrapolating the
linear currents obtained during voltage steps in more negative potential regions (
100 to
80 mV), where no voltage-activated currents were seen. Current amplitudes were small enough that the
series resistance error was <5 mV. Analog compensation was applied to
attenuate capacitive current transients and to estimate cell
capacitances, a measure of total cell plasma membrane area. Membrane
currents were measured with an Axopatch 1-D patch clamp (Axon
Instruments, Foster City, CA) and filtered at 1 kHz. The currents were
digitized and stored directly to disk (DigiData 1200 Interface, Axon
Instruments). Data analysis was performed with pClamp 6.0.3 software
(Axon Instruments).
Morphometric analysis of cells. Light-microscopic images of granulosa cells were collected from at least five identical locations of each incubation well or coverslip on an inverted Nikon microscope (20× objective) and stored. The outlines of individual cells were traced, and the parameters of mean surface area covered by each cell, cell perimeter, and circularity were determined with Optimas 6.0 Software (Bothell, WA). Higher estimates of circularity (which is independent of size) were associated with greater irregularity of cell profile. A perfect circle has a circularity of 12.
Determination of cell viability. Granulosa cells were incubated in 24-well culture plates precoated with solubilized basal lamina. Uncoated wells served as controls. After the removal of the serum-free M199 incubation medium, the cells were detached with trypsin (0.4 mg/ml) containing Ca2+ and Mg2+-free HBSS. The cells were washed two times with Ca2+-Mg2+-free HBSS, and viability was estimated with the trypan blue (0.1%) exclusion technique.
Data analysis. The data were analyzed by ANOVA followed by the post hoc Tukey test to determine significant differences among treatment means. A two-tailed t-test was performed where applicable. Differences at P < 0.05 were considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Collagenase-dispersed differentiated granulosa cells obtained from
the F1 of hen ovary were incubated in serum-free medium for 1-24 h
on tissue culture-treated plastic substratum (plastic substratum).
Patch clamp experiments were conducted in the conventional whole cell
configuration, with conditions under which primarily Ca2+
currents can be monitored. With a holding potential of 80 mV and step
commands between
100 and +80 mV, inward currents were recorded in the
differentiated granulosa cells. In ~32% of the cells studied, a fast
activating and inactivating inward (transient) current was recorded
(Fig. 1). The current was activated at
40 mV and required 8-13 ms to reach peak amplitude. In 52% of
cells, a slowly inactivating, long-lasting current was superimposed on the fast activating/inactivating one (Fig.
2); this current remained steady, not
returning to basal levels within the duration of the test pulse. The
remainder of the cells (~15%) did not express inward currents under
the present experimental conditions.
|
|
Characterization of the Ca2+
currents.
The types of Ca2+ currents expressed by the granulosa
cells were determined by modifying the holding potential and applying known Ca2+ channel blockers. When the holding potential was
changed from 80 to
50 mV, the amplitude of the fast
activating/inactivating (transient) current decreased dramatically
(Figs. 1B and 2B); this is an indication that it
is a T-type Ca2+ current. Indeed, the application of the
T-type Ca2+ channel blocker flunarizine (20 µM)
suppressed the fast activating transient current (Fig. 1C),
supporting the view that it is a T-type Ca2+ current. In
cells that expressed both transient and long-lasting inward currents,
flunarizine (20 µM) blocked the transient current component but not
the long-lasting current. The remaining current (long-lasting current)
had activation/inactivation kinetics that are reminiscent of the L-type
Ca2+ current (see Fig. 2B). The long-lasting
current was suppressed by the L-type Ca2+ channel blocker
nifedipine (5 µM) (Fig. 2C), demonstrating that it is an
L-type Ca2+ current. In additional experiments, nifedipine
blocked the long-lasting current component (Fig.
3B) but not the fast current
component that could be blocked by flunarizine (Fig. 3C).
The peak of the transient current was regarded as the amplitude of
T-type current. The amplitude of the L-type current was measured at the
end of the step potential.
|
Effect of intact basal lamina on T- and L-type
Ca2+ currents and membrane capacitance.
Differentiated granulosa cells incubated on intact basal lamina for
24 h in serum-free M199 assumed spherical shape, whereas those
incubated on plastic substratum in control dishes became highly
flattened (see Fig. 7). The T-type Ca2+ current was
suppressed in differentiated granulosa cells incubated on basal lamina
(Fig. 4B). Similarly, the
L-type Ca2+ current was inhibited in cells incubated on
basal lamina (Fig. 4E). The current-voltage relation of T-
and L-type Ca2+ currents demonstrates a reduction in
current amplitudes at several voltages (Fig. 4, C and
F). The amplitude of the T-type Ca2+ current
(Fig. 5A) or L-type
Ca2+ current (Fig. 5D) was significantly
(P < 0.05) greater for cells incubated on plastic than
for cells incubated on basal lamina. When the T- and L-type
Ca2+ current amplitudes were normalized to plasma membrane
area, the current densities in granulosa cells incubated on basal
lamina were significantly (P < 0.05) smaller than
those of cells incubated on plastic (Fig. 5, B and
E). Membrane capacitance, an indirect measure of total
plasma membrane area (and directly proportional to membrane area), was
significantly (P < 0.05) smaller for cells incubated
on intact basal lamina than for the control cells incubated on plastic
(Fig. 5C).
|
|
Time course of effect of intact basal lamina on T- and L-type
Ca2+ currents.
Both T- and L-type Ca2+ currents could be recorded within
1 h of incubation of differentiated granulosa cells in serum-free medium; the T- and L-type Ca2+ currents remained expressed
throughout 24 h of incubation (Fig. 6). Basal lamina suppressed significantly
(P < 0.05) both T- and L-type Ca2+
currents after 24-h incubation; however, it had marginal (but nonsignificant) inhibitory effects on the currents after 1-12 h of
incubation (Fig. 6).
|
Time course of effect of intact basal lamina on cell shape.
Time course experiments revealed that intact basal lamina caused
granulosa cells to become rounded within 1 h of incubation in
serum-free medium. By comparison, granulosa cells incubated on plastic
became flat. The morphometric parameters of granulosa cells cultured on
intact basal lamina or plastic are shown in Fig.
7; within 60 min of incubation, the mean
area occupied by cells incubated directly on the basal lamina was
significantly (P < 0.05) less than that of cells
incubated on plastic (Fig. 7A). Similarly, the morphometric
parameter of perimeter was significantly (P < 0.05)
lower after 60 min of incubation on basal lamina (Fig. 7B).
The circularity of cells was reduced (P < 0.05) by
basal lamina after 24 h of incubation (Fig. 7C).
|
Influence of storage on the effect of intact basal lamina on
membrane capacitance and T- and L-type
Ca2+ currents.
Membrane capacitance and T- and L-type Ca2+ currents were
recorded in differentiated granulosa cells incubated in intact basal lamina-containing culture dishes that had been stored for 12 mo or
longer; the results are shown in Fig. 8.
The membrane capacitance of cells incubated on intact basal lamina
stored for 12 mo was significantly (P < 0.05) smaller
than that in the cells incubated on plastic (Fig. 8A). Also,
the density of the T- and L-type Ca2+ currents in control
cells was significantly (P < 0.05) larger than those
of cells incubated on basal lamina stored for 12 mo (Fig. 8,
A and B).
|
Relation between membrane capacitance (cell shape) and T- and
L-type Ca2+ currents.
Because of the apparently consistent relationship between the
expression of T- and L-type Ca2+ currents and membrane
capacitance (cell shape) in response to basal lamina, correlative
changes in these two parameters in individual cells were estimated. The
amplitudes or densities of the Ca2+ currents of granulosa
cells incubated on plastic and basal lamina were plotted vs. their
respective membrane capacitances (Fig. 9). The slopes of the linear regressions
were not different from zero (P > 0.05), demonstrating
that no correlation existed between the amplitude or density of the T-
and L-type Ca2+ currents and the membrane capacitance (cell
shape) in granulosa cells incubated on plastic or on basal lamina.
Moreover, the relative changes in amplitudes of the T- and L-type
Ca2+ currents (50-80%) in cells incubated on basal
lamina were between 2.7- and 4.2-fold greater than the change in
membrane capacitance (19%) (see Fig. 5). This suggested that changes
in membrane capacitance (associated with cell shape) alone cannot
account for the observed effects of basal lamina on the T- and L-type
Ca2+ currents.
|
Effect of solubilized basal lamina on T- and L-type
Ca2+ currents.
In additional experiments, the effects of solubilized basal lamina on
T- and L-type Ca2+ currents were assessed. Differentiated
granulosa cells were incubated on coverslips precoated with solubilized
basal lamina (5 µg/cm2) (see MATERIALS AND
METHODS). Similar to observations made for granulosa cells
incubated on intact basal lamina, granulosa cells incubated on
coverslips precoated with solubilized basal lamina have reduced
membrane capacitance and T- and L-type Ca2+ current density
(Fig. 10).
|
Effects of fibronectin, laminin, or type IV collagen on T- and
L-type Ca2+ currents.
Experiments were conducted to determine the effects of extracellular
matrix proteins that are components of basement membranes on T- and
L-type Ca2+ currents. Differentiated granulosa cells were
incubated in culture wells precoated with fibronectin (5 µg/cm2), laminin (2 µg/cm2), type IV
collagen (5 µg/cm2), or solubilized basal lamina (5 µg/cm2). Similar to observations made for granulosa cells
incubated on solubilized basal lamina, granulosa cells incubated on
fibronectin, laminin, or collagen IV have reduced membrane capacitance
and T- and L-type Ca2+ current density (Fig.
11).
|
Effects of cycloheximide and actinomycin D on the
Ca2+ currents.
The effects of the protein synthesis inhibitor cycloheximide and
transcription inhibitor actinomycin D on the T- and L-type Ca2+ currents were tested. Differentiated granulosa cells
were incubated on plastic and intact basal lamina for 24 h, and
membrane currents were recorded. Cycloheximide or actinomycin D had no
appreciable effect on the expression of the Ca2+ currents
in granulosa cells incubated on plastic; similarly, the inhibitors had
no significant effect on Ca2+ currents in granulosa cells
incubated on basal lamina (Fig. 12).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of the present study show that basal lamina of the
chicken ovarian follicle can regulate T- and L-type Ca2+
currents in differentiated chicken granulosa cells. The ability of the
basal lamina to suppress Ca2+ currents was not affected by
solubilization, because, similar to the intact form, the solubilized
basal lamina (containing components with molecular mass >3 kDa)
suppressed the T- and L-type Ca2+ currents in
differentiated granulosa cells. The concentration of solubilized basal
lamina used in the present study was shown to significantly regulate
progesterone production in chicken granulosa cells (5). An
interesting finding of the present study was that the storage of basal
lamina for 12 mo at 4°C did not affect its ability to regulate the
Ca2+ currents. In relation to this observation is the
recent finding that basal lamina stored for
18 mo regulated the shape
of granulosa cells to a similar extent as freshly prepared ones
(7). The present results confirm an earlier report that
chicken granulosa cells express T-and L-type voltage-dependent
Ca2+ channels (58). The present results also
confirm reports that chicken (14, 45, 57, 58, 65) and
porcine (38) granulosa cells express whole cell T-type
Ca2+ currents.
How did the basal lamina attenuate the expression of Ca2+ currents? The possibility remains that basal lamina inhibited the activation of existing Ca2+ channels; however, it is also possible that basal lamina reduced the number of Ca2+ channel proteins or suppressed the synthesis of Ca2+ channel proteins. The observation that the exposure of differentiated granulosa cells (incubated on plastic or basal lamina for 24 h) to cycloheximide or actinomycin D did not result in a change in the effects of basal lamina on the Ca2+ currents indicates, perhaps, that the actions of basal lamina monitored here did not directly involve gene transcription or protein synthesis. The mechanisms whereby basal lamina exerted its effects will be determined in the future.
The observations that known components of basement membranes, fibronectin, laminin, and type IV collagen suppressed both T- and L-type Ca2+ currents (in the present study) suggest that the actions of intact basal lamina are mediated in part by these matrix proteins. It is noteworthy that progesterone production by rat granulosa cells incubated in serum-free medium for 24 h in laminin-coated or fibronectin-coated wells decreased significantly (1). Moreover, steroidogenesis was suppressed in differentiated human granulosa cells cultured in laminin-coated wells (26). Other investigators have shown that extracellular matrix proteins, especially those of basement membrane origin, have been shown to regulate ion transport in various types of cells. For example, inward whole cell Na+ currents were significantly reduced in rat adult alveolar type II cells cultured on fibronectin-coated coverslips for 48 h (71). Also, rat adult alveolar type II cells cultured on human amniotic basement membrane had smaller short-circuit currents than cells cultured on collagen substrate (20). Furthermore, Matrigel (reconstituted basement membrane) modulated the K+ channel density in fetal rat alveolar type II cells (41).
Because granulosa cells are exposed to the basal lamina as a unit in
vivo (but not individual components in isolation), the effect of basal
lamina was examined as a unit (in the present study) with the
presumption that the outcome would be the result of combined effects of
different components of the matrix material. Although, in the present
study, the nature and characteristics of the components of basal lamina
that regulated the inward currents and their mechanisms of action are
unknown, some of the components are likely to stimulate cellular
processes that other components suppress, whereas some components would
be without effect. Therefore, the actions of the individual components
of basal lamina may be synergistic, additive, antagonistic, or neutral,
as the case may be. It was shown recently that several components of
basal lamina of chicken ovarian follicle reacted positively to
antibodies raised against extracellular matrix proteins (type IV
collagen, laminin, entactic, heparan sulfate proteoglycan, and
fibronectin), growth factors [epidermal (EGF), platelet-derived
(PDGF), basic and acidic fibroblast (FGF), insulin-like (IGF),
transforming (TGF-, TGF-
)], cytokines, matrix
metalloproteinases, and their tissue inhibitors (8). The
possibility remained that these bioactive components of basal lamina
acted concomitantly to regulate the Ca2+-carried inward
currents in the present studies. Indeed, the actions of integrins
(receptors of extracellular matrix proteins) may synergize or overlap
with those of growth factors. [Integrins are a large family of
heterodimeric transmembrane proteins with different
- and
-subunits that function as extracellular matrix adhesion receptors
(33)]. Integrins can activate signaling pathways known to
be regulated by growth factors. For example, integrins have been shown
to cooperate with growth factors to increase signaling for mitogenic
processes (40, 48, 64). Similarly, growth factors EGF,
PDGF-BB, or basic FGF cooperated with integrins (ligand occupied and
aggregated) to enhance the transient activation of the extracellular
signal-regulated kinase class of mitogen-activated protein (MAP) kinase
(48). In addition, EGF, PDGF, or FGF also synergized with
integrins (ligand occupied and aggregated) to enhance tyrosine
phosphorylation of the growth factor receptors (48).
Integrins activated Shc, which is known to link several growth factor-activated pathways (70). In certain cases,
integrins and growth factors cooperated to increase cell proliferation
(40, 48, 64). Integrin-mediated adhesion (cell anchorage)
was shown to be necessary for growth factor (EGF, PDGF) activation of
the MAP kinase cascade (40). In addition,
integrin-mediated adhesion was required for PDGF-stimulated
phosphatidylinositol bisphosphate hydrolysis (44).
Integrins activated the Ras-Raf-MAP kinase pathway, protein kinase C,
and phosphatidylinositol 3'-kinase (13, 16, 59).
Earlier studies showed that progesterone synthesis in granulosa cells of several species was reduced in Ca2+-deficient incubation media (6, 22, 61, 63), and a recent study demonstrated that the abundance of messenger RNA of cytochrome P450scc (the enzyme that catalyzes the metabolism of cholesterol to pregnenolone) in porcine granulosa cells was reduced in Ca2+-deficient medium (37). Therefore, one of the mechanisms by which Ca2+ modulates steroid hormone synthesis is the increase in the transactivation of the cytochrome P450scc gene (37). Because, in granulosa cells, the suppression of uptake of extracellular Ca2+ resulted in the attenuation of progesterone production (6, 22, 61, 63) and transcription of the cytochrome P450scc gene, and because basal lamina suppressed inward Ca2+ current in differentiated granulosa cells (in the present study), it would be expected that basal lamina would decrease progesterone production in differentiated chicken granulosa cells. Indeed, chicken ovarian basal lamina suppressed progesterone synthesis in differentiated chicken granulosa cells in vitro (7). It is noteworthy that the suppression of steroidogenesis in differentiated granulosa cells by solid basement membrane or extracellular matrices is not limited to avian granulosa cells. Notably, Aten et al. (9) observed that rat granulosa cells incubated on Matrigel matrix, basement membrane reconstituted from extracts of Engelbreth-Holm-Swarm tumor, produced less progesterone than those incubated on plastic. In addition, progesterone production by rat granulosa cells in laminin-coated wells decreased (1). In other studies, when human granulosa cells retrieved from patients undergoing in vitro fertilization were cultured in laminin-coated wells, steroidogenesis was suppressed (26). Similarly, human chorionic gonadotropin-induced progesterone synthesis was attenuated in laminin-coated dishes (26). Therefore, reports in the literature indicate that the incubation of granulosa cells in dishes precoated with reconstituted basement membrane, components of basement membranes, or basal lamina results in the reduction of steroid hormone synthesis. The relation between an extracellular matrix-induced rounded shape and steroidogenesis in granulosa cells was reported (for review see Ref. 2). It appears that the nature of the relation between basement membrane protein-induced rounded shape and steroid hormone synthesis is influenced by factors such as the state of granulosa cell differentiation and the form of the matrix protein (whether solid or liquid) among others (1, 2, 7, 9, 26).
In the present study, basal lamina prevented the spreading/stretching of granulosa cells and reduced their membrane capacitance (a measure of plasma membrane surface area). It was reported that chicken granulosa cells, especially the differentiated ones, synthesize and deposit large quantities of fibronectin in serum-free culture (4, 18). The morphology of granulosa cells incubated on tissue culture-treated plastic substratum was likely caused by integrin-mediated adhesion of cells to newly deposited (endogenous) extracellular matrix, and not by direct interaction with plastic. It is therefore possible that, in granulosa cells incubated on basal lamina, the interaction of cells with deposited endogenous extracellular matrix was prevented or attenuated, leading to the observed changes in cell adhesion, morphology, and function (6, 7, and present study). As such, the spreading on the tissue culture-treated plastic substratum probably reflects greater adhesion of granulosa cells to deposited extracellular matrix, such as fibronectin, than to cells incubated on basal lamina; that is, the granulosa cells spread less efficiently on basal lamina; consequently they became rounded (Ref. 7 and the present study).
It is known that mechanical force imposed on cells by stretch, gravity, hemodynamic (shear) stresses, and movement can influence cellular behavior and function. In tissue culture models, strong traction forces are exerted by cells (21, 31), and the spreading/stretching of cells can generate intracellular signals to regulate cell functions (15). The attachment of a cell to the extracellular matrix, via integrins, could also exert mechanical force (through tugging of the integrins) (45). This integrin-mediated force can be transmitted throughout a tensionally integrated cell and induce changes in chemical signaling, the organization of intracellular organelles, and reorganization (stiffness) of the cytoskeleton and nucleus, thereby regulating various physiological processes in the cell (34-36, 43, 60, 67-69). Thus the extracellular matrix (via local mechanical tension on cells) could regulate cell function by mechanically stabilizing the lattice of cytoskeleton and nucleus (43). Whether or not basal lamina can cause the reorganization of the cytoskeleton and intracellular organelles remains to be determined.
After the binding of extracellular matrix with integrin, a specialized cytoskeletal structure (known as focal adhesion complex) forms intracellularly at the site of integrin binding. The focal adhesion complex serves as a molecular bridge, and it mechanically couples integrins (integrin-extracellular matrix complex) to the actin cytoskeleton (11, 19, 47). This focal adhesion complex also participates in the direction of much of the signaling machinery of the cell (12, 16, 34, 35, 46, 54-56, 59). Therefore, focal adhesion complexes could integrate mechanical signals associated with changes in cell morphology with chemical signals generated by integrins and thereby regulate signaling downstream (36). Practically, any link that physically connects the transmembrane receptors to the cytoskeleton can mediate the transmission of mechanical force across the cell membrane. The focal adhesion complex can and does serve as a molecular link between the transmembrane integrins to the cytoskeleton (11). Integrins, therefore, could serve as mechanotransducers. It was shown that mechanical stretch activated stretch-sensitive Ca2+ channels that mediated the influx of extracellular Ca2+ in human umbilical endothelial cells (50), fibroblasts (30), and fish keratocytes (39). The roles of integrins in the actions of basal lamina in avian granulosa cells are yet to be determined.
In summary, the unique anatomic structure of the avian ovarian follicle enabled the isolation of pure and intact basal lamina (basement membrane) and its associated granulosa cells and provided the opportunity to study the effect of basal lamina on transmembrane ion currents in the granulosa cells in a homologous system. Both intact basal lamina and its solubilized form suppressed inward Ca2+ currents in differentiated granulosa cells. The results may explain (partly) the effect of basal lamina on the metabolic functions of differentiated avian granulosa cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Michael Kinch and Riyi Shi for their comments on the manuscript.
![]() |
FOOTNOTES |
---|
This work was supported by funds from Purdue University School of Veterinary Medicine. W. Qin was supported by a Purdue Research Foundation research assistantship.
Present address of S. G. Rane: Fujisawa Research Institute of America, Northwestern University/Evanston Research Park, 1801 Maple Ave., Evanston, IL 60201.
Address for reprint requests and other correspondence: E. K. Asem, Dept. of Basic Medical Sciences, School of Veterinary Medicine, Purdue Univ., 1246 Lynn Hall, West Lafayette, IN 47907-1246 (E-mail: asem{at}vet.purdue.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 June 2001; accepted in final form 30 August 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aharoni, D,
Meiri I,
Atzmon R,
Vlodavsky I,
and
Amsterdam A.
Differential effect of components of the extracellular matrix on differentiation and apoptosis.
Curr Biol
7:
43-51,
1997[ISI][Medline].
2.
Amsterdam, A,
and
Rotmensch S.
Structure-function relationships during granulosa cell differentiation.
Endocr Rev
8:
309-330,
1987[ISI][Medline].
3.
Amsterdam, A,
Rotmensch S,
Furman A,
Venter EA,
and
Vlodavsky I.
Synergistic effect of human chorionic gonadotropin and extracellular matrix on in vitro differentiation and gap junction formation.
Endocrinology
124:
1956-1964,
1989[Abstract].
4.
Asem, EK,
Conkright MD,
and
Novero RP.
Progesterone stimulates fibronectin production by chicken granulosa cells in vitro.
Eur J Endocrinol
130:
159-165,
1994[ISI][Medline].
5.
Asem, EK,
Feng S,
Stingley-Salazar SR,
Turek JJ,
Peter AT,
and
Robinson JP.
Basal lamina of avian ovarian follicle: influence on morphology of granulosa cells in-vitro.
Comp Biochem Physiol
125C:
189-201,
2000[ISI].
6.
Asem, EK,
and
Hertelendy F.
Role of calcium in luteinizing hormone-induced progesterone and cyclic AMP production in granulosa cells of the hen (Gallus domesticus).
Gen Comp Endocrinol
62:
120-128,
1986[ISI][Medline].
7.
Asem, EK,
Stingley-Salazar SR,
Robinson JP,
and
Turek JJ.
Effect of basal lamina on progesterone production by chicken granulosa cells in vitroinfluence of follicular development.
Comp Biochem Physiol
125C:
233-244,
2000[ISI].
8.
Asem, EK,
Stingley-Salazar SR,
Robinson JP,
and
Turek JJ.
Identification of some components of basal lamina of avian ovarian follicle.
Poult Sci
79:
589-601,
2000[ISI][Medline].
9.
Aten, RF,
Kolodecik TR,
and
Behrman HR.
A cell adhesion receptor antiserum abolishes, whereas laminin and fibronectin glycoprotein components of extracellular matrix promote, luteinization of cultured rat granulosa cells.
Endocrinology
136:
1753-1758,
1995[Abstract].
10.
Bakst, MR.
Scanning electron microscopy of hen granulosa cells before and after ovulation.
Scan Electron Microsc
3:
307-312,
1979.
11.
Burridge, K,
Fath K,
Kelley T,
Nuckolls G,
and
Turner C.
Focal adhesions: transmembrane junctions beween the extracellular matrix and the cytoskeleton.
Annu Rev Cell Biol
4:
487-525,
1988[ISI].
12.
Burridge, K,
Turner C,
and
Romer L.
Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly.
J Cell Biol
119:
893-904,
1992[Abstract].
13.
Chen, Q,
Kinch MS,
Lin TH,
Burridge K,
and
Juliano RL.
Integrin-mediated cell adhesion activates mitogen-activated protein kinases.
J Biol Chem
269:
26602-26605,
1994
14.
Chiang, M,
Strong JA,
and
Asem EK.
Bovine serum albumin increases Ca2+ currents in chicken ovarian granulosa cells.
Mol Cell Endocrinol
94:
27-36,
1993[ISI][Medline].
15.
Chiquet, M.
Regulation of extracellular matrix gene expression by mechanical stress.
Matrix Biol
18:
417-426,
1999[ISI][Medline].
16.
Clarke, EA,
and
Brugge JS.
Integrins and signal transduction: the road taken.
Science
268:
233-238,
1995[ISI][Medline].
17.
Conkright, MD,
and
Asem EK.
Intracrine role of progesterone in fibronectin production and deposition by chicken ovarian granulosa cells in vitro: effect of extracellular calcium.
Biol Reprod
52:
683-689,
1995[Abstract].
18.
Conkright, MD,
and
Asem EK.
Intracrine role of progesterone in follicle-stimulating hormone- and cyclic adenosine 3',5'-monophosphate-induced fibronectin production and deposition by chicken granulosa cells: influence of follicular development.
Endocrinology
136:
2641-2651,
1995[Abstract].
19.
Craig, SW,
and
Johnson RP.
Assembly of focal adhesions: progress, paradigms, and portents.
Curr Opin Cell Biol
8:
74-85,
1996[ISI][Medline].
20.
Cott, GR.
Modulation of bioelectric properties across alveolar type II cells by substratum.
Am J Physiol Cell Physiol
257:
C678-C688,
1989
21.
Danowski, BA,
and
Harris AK.
Changes in fibroblast contractility, morphology, and adhesion in response to a phorbol ester tumor promoter.
Exp Cell Res
177:
47-59,
1988[ISI][Medline].
22.
Eckstein, N,
Eshel A,
Eli Y,
Ayalon D,
and
Naor Z.
Calcium-dependent actions of gonadotropin releasing hormone agonist and luteinizing hormone upon cyclic AMP and progesterone production in rat ovarian granulosa cells.
Mol Cell Endocrinol
47:
91-98,
1986[ISI][Medline].
23.
Flores, JA,
Aguirre C,
and
Veldhuis JD.
Luteinizing hormone stimulates both intracellular calcium ion ([Ca2+]i) mobilization and transmembrane cation influx in single ovarian (granulosa) cells: recruitment as a cellular mechanism of LH-[Ca2+]i dose-response.
Endocrinology
139:
3606-3612,
1998
24.
Flores, JA,
Leong DA,
and
Veldhuis JD.
Is the calcium signal induced by follicle-stimulating hormone in swine granulosa cells mediated by adenosine cyclic 3',5'-monophosphate-dependent protein kinase?
Endocrinology
130:
1862-1866,
1992[Abstract].
25.
Flores, JA,
Veldhuis JD,
and
Leong DA.
Follicle-stimulating hormone evokes an increase in intracellular free calcium ion concentrations in single ovarian (granulosa) cells.
Endocrinology
127:
3172-3179,
1990[Abstract].
26.
Fujiwara, H,
Honda T,
Ueda M,
Nakamura K,
Yamada S,
Maeda M,
and
Mori T.
Laminin suppresses progesterone production by human luteinizing granulosa cells via interaction with integrin 6
1.
J Clin Endocrinol
82:
2122-2128,
1997
27.
Furman, A,
Rotmensch S,
Dor J,
Venter A,
Mashiach S,
Vlodavsky I,
and
Amsterdam A.
Culture of human granulosa cells from an in vitro fertilization program: effects of extracellular matrix on morphology and cyclic adenosine 3',5' monophosphate production.
Fertil Steril
46:
514-517,
1986[ISI][Medline].
28.
Furman, A,
Rotmensch S,
Kohen F,
Mashiach S,
and
Amsterdam A.
Regulation of rat granulosa cell differentiation by extracellular matrix produced by bovine corneal endothelial cells.
Endocrinology
118:
1878-1885,
1986[Abstract].
29.
Gilbert, AB,
Evans AJ,
Perry MM,
and
Davidson MH.
A method for separating the granulosa cells, the basal lamina and the theca of the preoculatory ovarian follicle of the domestic fowl (Gallus domesticus).
J Reprod Fertil
50:
179-181,
1977[Medline].
30.
Glogauer, M,
Ferrier J,
and
McCulloch CAG
Magnetic fields applied to collagen-coated ferric oxide beads induce stretch-activated Ca2+ influx in fibroblasts.
Am J Physiol Cell Physiol
269:
C1093-C1104,
1995
31.
Harris, AK,
Stopak D,
and
Wild P.
Fibroblast traction as a mechanism for collagen morphogenesis.
Nature
290:
249-251,
1981[ISI][Medline].
32.
Hertelendy, F,
Nemecz G,
and
Molnar M.
Influence of follicular maturation on luteinizing hormone and guanosine 5'-O-thiotriophosphate-promoted breakdown of phosphoinositides and calcium mobilization in chicken granulosa cells.
Biol Reprod
40:
1144-1151,
1989[Abstract].
33.
Hynes, RO.
Integrins: versatility, modulation and signalling in cell adhesion.
Cell
69:
11-29,
1992[ISI][Medline].
34.
Ingber, DE.
The riddle of morphogenesis: a question of solution chemistry or molecular cell engineering?.
Cell
75:
1249-1252,
1993[ISI][Medline].
35.
Ingber, DE.
Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton.
J Cell Sci
104:
613-627,
1993
36.
Ingber, DE.
Tensegrity: the architectural basis of cellular mechanotransduction.
Annu Rev Physiol
59:
575-599,
1997[ISI][Medline].
37.
Jayes, FCL,
Day RN,
Garmey JC,
Urban RJ,
Zhang G,
and
Veldhuis JD.
Calcium ions positively modulate follicle-stimulating hormone- and exogenous cyclic 3',5'-adenosine monophosphate-driven transcription of the cytochrome P450scc gene in porcine granulosa cells.
Endocrinology
141:
2377-2384,
2000
38.
Kusaka, M,
Tohse N,
Nakaya H,
Tanaka T,
Kanno M,
and
Fujimoto S.
Membrane currents of porcine granulosa cells in primary culture: characterization and effects of luteinizing hormone.
Biol Reprod
49:
95-103,
1993[Abstract].
39.
Lee, J,
Ishihara A,
Oxford G,
Johnson B,
and
Jacobson K.
Regulation of cell movement is mediated by stretch-activated calcium channels.
Nature
400:
382-386,
1999[ISI][Medline].
40.
Lin, TH,
Chen Q,
Howe A,
and
Juliano RL.
Cell anchorage permits efficient signal transduction between ras and its downstream kinases.
J Biol Chem
272:
8849-8852,
1997
41.
Liu, S,
and
Mautone AJ.
Whole cell potassium currents in fetal rat alveolar type II cells cultured on Matrigel matrix.
Am J Physiol Lung Cell Mol Physiol
270:
L577-L586,
1996
42.
Luckhoff, A,
and
Clapham DE.
Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca2+-permeable channel.
Nature
355:
356-358,
1992[ISI][Medline].
43.
Maniotis, A,
Chen CS,
and
Ingber DE.
Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure.
Proc Natl Acad Sci USA
94:
849-854,
1997
44.
McNamee, HP,
Ingber DE,
and
Schwartz MA.
Adhesion to fibronection stimulates inositol lipid synthesis and enhances PDGF-induced inositol lipid breakdown.
J Cell Biol
121:
673-678,
1993[Abstract].
45.
Mealing, G,
Morley P,
Whitfield JF,
Tsang BK,
and
Schwartz JL.
Granulosa cells have calcium-dependent action potentials and a calcium-dependent chloride conductance.
Pflügers Arch
428:
307-314,
1994[ISI][Medline].
46.
Miyamoto, S,
Akiyama SK,
and
Yamada KM.
Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function.
Science
267:
883-885,
1995[ISI][Medline].
47.
Miyamoto, S,
Teramoto H,
Coso OA,
Gutkind JS,
Burbelo PD,
Akiyama SK,
and
Yamada KM.
Integrin function: molecular hierarchies of cytoskeletal and signaling molecules.
J Cell Biol
131:
791-805,
1995[Abstract].
48.
Miyamoto, S,
Teramoto H,
Gutkind JS,
and
Yamada KM.
Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors.
J Cell Biol
135:
1633-1642,
1997[Abstract].
49.
Morley, P,
Tsang BK,
Whitfield JF,
and
Schwartz JL.
The effect of muscarinic cholinergic agonists on intracellular calcium and progesterone production by chicken granulosa cells.
Endocrinology
130:
663-670,
1992[Abstract].
50.
Naruse, K,
and
Sokabe M.
Involvement of stretch-activated ion channels in Ca2+ mobilization to mechanical stretch in endothelial cells.
Am J Physiol Cell Physiol
264:
C1037-C1044,
1993
51.
Novero, RP,
and
Asem EK.
Follicle stimulating hormone-enhanced fibronectin production by chicken granulosa cells is influenced by follicular development.
Poult Sci
72:
709-721,
1993[ISI][Medline].
52.
Perry, MM,
Gilbert AB,
and
Evans AJ.
Electron microscopic observations on the ovarian follicle of the domestic fowl.
Am J Anat
23:
1-35,
1978.
53.
Perry, MM,
Gilbert AB,
and
Evans AJ.
Electron microscope observations on the ovarian follicle of the domestic fowl during the rapid growth phase.
J Anat
125:
481-497,
1978[ISI][Medline].
54.
Plopper, GE,
McNamee HP,
Dike LE,
Bojanowski K,
and
Ingber DE.
Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex.
Mol Biol Cell
6:
1349-1365,
1995[Abstract].
55.
Richardson, A,
and
Parsons T.
A mechanism for regulation of the adhesion-associated protein tyrosine kinase pp125FAK.
Nature
380:
538-540,
1996[ISI][Medline].
56.
Schlaepfer, DD,
and
Hunter T.
Integrin signaling and tyrosine phosphorylation: just the FAKs?
Trend Cell Biol
8:
151-157,
1998[ISI][Medline].
57.
Schwartz, JL,
Asem EK,
Mealing GAR,
Tsang BK,
Whitfield JF,
Rousseau E,
and
Payet MD.
T- and L-calcium channels in steroid-producing chicken granulosa cells in primary culture.
Endocrinology
125:
1973-1982,
1989[Abstract].
58.
Schwartz, JL,
Mealing GAR,
Asem EK,
Tsang BK,
and
Whitfield JF.
Ionic currents in avian granulosa cells.
FEBS Lett
241:
169-172,
1988[ISI][Medline].
59.
Schwartz, MA,
Schaller MD,
and
Ginsberg MH.
Integrins: emerging paradigms of signal transduction.
Annu Rev Cell Dev Biol
11:
549-599,
1995[ISI][Medline].
60.
Stamenovic, D,
Fredberg JJ,
Wang N,
Butler JP,
and
Ingber DE.
A microstructural approach to cytoskeletal mechanics based on tensegrity.
J Theor Biol
181:
125-136,
1996[ISI][Medline].
61.
Tsang, BK,
and
Carnegie JA.
Calcium requirement in the gonadotropic regulation of rat granulosa cell progesterone production.
Endocrinology
113:
763-769,
1983[Abstract].
62.
Veldhuis, JD.
Mechanisms subserving hormone action in the ovary: role of calcium ions as assessed by steady state calcium exchange in cultured swine granulosa cells.
Endocrinology
120:
445-229,
1987[Abstract].
63.
Veldhuis, JD,
Klase PA,
Demers LM,
and
Chafouleas JG.
Mechanisms subserving calcium's modulation of luteinizing hormone action in isolated swine granulosa cells.
Endocrinology
14:
441-449,
1984.
64.
Vuori, K,
and
Ruoslahti E.
Association of insulin receptor substrate-1 with integrins.
Science
266:
1576-1578,
1994[ISI][Medline].
65.
Wan, X,
Desilets M,
Soboloff J,
Morris C,
and
Tsang BK.
Muscarinic activation inhibits T-type Ca current in hen granulosa cells.
Endocrinology
137:
2514-2521,
1996[Abstract].
66.
Wang, J,
Baimbridge KG,
and
Leung PCK
Changes in cytosolic free calcium ion concentration in individual rat granulosa cells: effect of luteinizing hormone releasing hormone.
Endocrinology
124:
1912-1921,
1989[Abstract].
67.
Wang, H,
Butler JP,
and
Ingber DE.
Mechanotransduction across the cell surface and through the cytoskeleton.
Science
260:
1124-1127,
1993[ISI][Medline].
68.
Wang, H,
and
Ingber DE.
Control of cytoskeletal mechanics by extracellular matrix, cell shape, and mechanical tension.
Biophys J
66:
2181-2189,
1994[Abstract].
69.
Wang, N,
and
Ingber DE.
Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry.
Biochem Cell Biol
73:
327-335,
1995[ISI][Medline].
70.
Wary, KK,
Mainiero F,
Isakoff SJ,
Marcantonio EE,
and
Giancotti F.
The adaptor protein Shc couples a class of integrins to the control of cell cycle progression.
Cell
87:
733-743,
1996[ISI][Medline].
71.
Yue, G,
Hu P,
Oh Y,
Jilling T,
Shoemaker RL,
Benos DJ,
Cragoe EJ, Jr,
and
Matalon S.
Culture-induced alterations in alveolar type II cell Na+ conductance.
Am J Physiol Cell Physiol
265:
C630-C640,
1993
72.
Yurchenco, PD,
and
Schittny JC.
Molecular architecture of basement membranes.
FASEB J
4:
1577-1590,
1990
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |