1 Department of Basic Medical Sciences, School of Veterinary Medicine, and 2 Department of Biological Sciences, Purdue University, West Lafayette, Indiana 49707
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ABSTRACT |
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Patch-clamp
experiments were conducted to study the effects of basal lamina
(basement membrane) of preovulatory chicken ovarian follicle on
membrane currents in differentiated chicken granulosa cells in a
homologous system. The membrane capacitance (measure of total membrane
area) was smaller in cells cultured on intact basal lamina than that of
control cells. The granulosa cells expressed outward and two inward
currents. A small fraction of the cells (3%) expressed only a
transient fast-activating and -inactivating inward current carried by
Ca2+. The majority of the cells, however, expressed a
slowly activating and inactivating inward current (carried by
Cl) that was superimposed on the transient
Ca2+ current. All cells expressed an outward current
characteristic of the delayed-rectifier K+ current. The
removal of extracellular Ca2+ led to elimination of the
slow inward Cl
current, indicating that it is a
Ca2+-dependent Cl
current. Both peak
amplitude and current density of the inward Cl
current
were significantly lower in cells cultured on freshly isolated intact
basal lamina (or basal lamina stored at 4°C for 12 mo) than those of
control cells; however, basal lamina had no significant effect on the
density of the outward current. Similar to the observations made for
intact basal lamina, solubilized basal lamina suppressed the inward
Cl
current in differentiated granulosa cells. These data
show that homologous basal lamina modulates a
Ca2+-dependent Cl
current in differentiated
granulosa cells. These findings provide a partial explanation for the
mechanisms that subserve the reported effects of basal lamina (basement
membrane) on the metabolic functions of differentiated granulosa cells.
basement membrane; patch clamp; chloride current; chicken; ovary; extracellular matrix; stretch-sensitive ion channels
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INTRODUCTION |
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BASEMENT MEMBRANES (basal laminae) are extracellular matrix sheets that compartmentalize tissues and act as physical barriers to macromolecules of body fluids. They influence cell shape and polarity and regulate physiological processes such as cell migration, growth, differentiation, and molecular filtration (21, 55). The components of basal laminae interact with cells that they support and transduce important signals to them via the integrin family of cell surface receptors (27).
Several investigators have provided evidence in support of the notion that basement membrane proteins modulate ovarian granulosa cell function. For example, basement membrane deposited in vitro by bovine corneal endothelial cells was shown to regulate steroidogenesis in rat and human granulosa cells (1-3, 23, 24). In addition, basement membrane proteins extracted from Engelbreth-Holm-Swarm tumor regulated progesterone synthesis in rat granulosa cells in vitro (10). Furthermore, basal lamina of avian ovarian follicle regulated progesterone synthesis in avian granulosa cells in a homologous system (7). Moreover, different types of extracellular matrix proteins such as collagen matrix have been shown to alter steroid hormone synthesis in rat and human granulosa cells in vitro (1, 12, 13).
Inorganic ions play significant roles in metabolic functions in ovarian
granulosa cells. Results of experiments conducted in vitro show that
steroidogenesis in mammalian and avian granulosa cells requires the
presence of Ca2+ (5, 20, 47, 48),
Na+ (32), and Cl
(37) in the incubation media. In addition, changes in
cytosolic levels of monovalent (K+ and Na+) and
divalent (Ca2+) cations are critical events in the
regulation of the functions of the ovarian granulosa cell
(31). It was demonstrated that inorganic ions enter or
leave granulosa cells through transmembrane channels (6, 16, 17,
29, 35, 36, 45, 46) or are carried by nonchannel transport
proteins (9, 32, 33).
Although basal lamina regulates granulosa cell functions, the signaling mechanisms that subserve its actions are unknown. Because transmembrane ion transport is required for steroid hormone synthesis in granulosa cells, it was hypothesized that basal lamina modulates membrane currents in these cells. Therefore, experiments were conducted to study the effects of basal lamina (as a unit) on transmembrane ion transport in granulosa cells. The combination of basal lamina and granulosa cells isolated from the preovulatory follicle of the chicken ovary was used as a model system to study the effects of basal lamina on granulosa cells in a homologous system. This homologous system was made possible by the anatomical structure of the avian ovarian follicle. In the mature avian ovarian follicle, the granulosa cell layer (membrana granulosa) consists of a single layer of cells located between the basal lamina and perivitelline layer (11, 13, 41, 42, 52); this arrangement made possible the isolation of intact basal lamina in hypotonic solution (4, 7).
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MATERIALS AND METHODS |
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Chemicals. HEPES, collagenase type IV, soybean trypsin inhibitor, BSA (fraction V), penicillin G, streptomycin, N-methyl-D-glucamine, fungizone, Trizma base, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic disodium salt (DIDS), tetraethylammonium chloride (TEA), nifedipine, actinomycin D, and cycloheximide were purchased from Sigma Chemical (St. Louis, MO). Medium 199 containing Hanks' salts was from GIBCO-BRL (Grand Island, NY). 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was purchased from Research Biochemicals International (Natick, MA). Flunarizine was obtained from Calbiochem (San Diego, CA).
Solutions. The currents were recorded under quasi-physiological conditions (high K+ in pipette and Na+ in bath solution). Unless noted otherwise, the pipette solution contained (in mM) 139 KCl, 5 NaCl, 3 ATPMg, 0.5 GTP, 10 HEPES, 0.1 EGTA, and 1 MgCl2 (pH 7.2). The bath solution was modified Hanks' balanced salt solution containing (in mM) 134.3 NaCl, 5.4 KCl, 2.5 CaCl2, 1.1 MgCl2, 5.6 glucose, and 10 HEPES (pH 7.4). In some cases, the bath solution contained 10 mM barium chloride instead of 2.5 mM CaCl2. Where required, Cs+ was substituted for K+ in the pipette solution and 10 mM TEA was included in the bath solution.
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-darkness 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 to the nearest 30 min (daily). Animals were injected with ketamine (50 mg/kg body wt) 10 min before being killed by cervical dislocation ~10-12 h before the expected time of ovulation of the largest preovulatory follicle (F1). The largest preovulatory follicle was removed and placed in ice-cold Hanks' salt solution containing (in mM) 140 NaCl, 5 KCl, 1.1 MgCl2, 2.5 CaCl2, 10 HEPES, and 5.6 glucose (pH 7.4). The theca and granulosa cell layers (membrana granulosa) were separated by the method of Gilbert et al. (25).
Isolation of intact basal lamina. Basal lamina was isolated as previously described (4, 7). Briefly, the granulosa cell layer obtained from the largest preovulatory follicle (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 hypoosmotically, and the basal lamina and perivitelline layer were separated. This basal lamina of the avian ovarian follicle preparation is an intact and complete basal lamina. The side of basal lamina that was in contact with granulosa cells in situ was designated as the "granulosa side," and the side in contact with theca tissue was designated as the "theca side."
Solubilization of basal lamina.
Basal laminae were solubilized in one step. They were placed in a
microfuge tube and solubilization buffer containing 6 M guanidine
hydrochloride and 50 mM Tris · HCl (pH 7.4), and 5% -mercaptoethanol was added (100 µl · basal
lamina
1 · follicle
1). 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) and allowed to dry in a laminar flow hood. Blank dishes served as controls. 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 aluminum 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). Coverslips that received vehicle only served as controls. The precoated coverslips were either used immediately or wrapped in aluminum foil and stored at 4°C. Tissue isolation, solubilization, dialysis, and preparation of coverslips were carried out under sterile conditions.
Enzymatic isolation of granulosa cells. Granulosa cells were dispersed in medium 199 containing 350 mg/l NaHCO3, 10 mM HEPES, 500,000 U/l collagenase, and 200 mg/l trypsin inhibitor at pH 7.4 (39). Cell viability, determined by the trypan blue exclusion method, was routinely >95%.
Cell culture. Collagenase-dispersed chicken granulosa cells were plated in intact basal lamina-containing dishes or in 35-mm dishes containing coverslips that were precoated with a total fraction of solubilized basal lamina. The cells were incubated for 24 h at 37°C in serum-free medium 199 containing 0.1% (wt/vol) BSA and 10 mM HEPES (pH 7.4; see Ref. 39).
Voltage clamp.
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 otherwise noted, 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. Junction potentials were corrected
manually on the amplifier. The bath solution and the reference
electrode were connected with a 3 M KCl-containing agar bridge. This
permitted recordings to be made without readjusting the junction
potential after the changing of bath solution. Analog compensation was
applied to attenuate capacitive current transients and to estimate cell
capacitances, a measure of the 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) and were analyzed with pCLAMP 6.0.3 software (Axon Instruments).
Data analysis. For current traces that were obtained with application of a 333-ms test pulse, the amplitudes of both inward and outward currents were measured at the end of the command pulse and used to plot current-voltage relationship curves. In experiments in which a 10.5-s test pulse was applied, the amplitude of the inward current was measured 250-800 ms after the onset of the command pulse. A t-test (2-tailed) was performed to compare the differences among treatment means and control values. Differences at P < 0.05 were considered significant.
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RESULTS |
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Freshly isolated differentiated granulosa cells (obtained from the
largest preovulatory chicken ovarian follicle) were incubated in
serum-free medium 199 for 24 h on plastic or glass coverslips and
studied with the conventional whole cell patch-clamp technique. With a
holding potential of 80 mV, depolarizing voltage steps between
60
and +80 mV activated outward and inward currents. In very few cells
(~3%) only a rapidly activating and inactivating inward current
(transient current) was activated (Fig.
1). In ~14% of cells, in addition to
the transient inward current, a large slowly activating and
inactivating inward current (slow current) could be activated as well
(Fig. 2). In the majority of cells, one
large inward current that appeared to have two components was observed
(Fig. 3).
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Examples of current traces recorded with a 333-ms command pulse,
holding potential of 80 mV, and depolarizing voltage steps between
60 and +80 mV are shown in Figs. 1A, 2A, and
3A. In Figs. 1-3, the insets show sections
of current traces obtained with a
40-mV voltage step presented on an
expanded scale. Figure 3A, inset, shows a current
trace that has two components, suggesting that the tracing is the
result of combinations of transient and slowly activating inward
currents. To determine the inactivation characteristics of the slowly
activating/inactivating current, the cells were activated with longer
command pulses (10.5-s command pulse from a holding potential of
80
mV to a depolarizing voltage step of
40 mV), and the results are
shown in Figs. 1B, 2B, and 3B. The
current traces shown in Figs. 1B, 2B, and
3B were recorded in cells from which currents in Figs.
1A, 2A, and 3A were recorded.
When observed together, the amplitude of the transient inward current
was always smaller than that of the slowly activating/inactivating current. The larger of the two inward currents could be elicited at
potentials between 60 and
50 mV, and it required 250-800 ms to
reach peak amplitude and 2-3 s to fully inactivate (Fig. 2,
A and B), whereas the fast-activating transient
inward current required 15-30 ms to reach a peak (Fig.
1A, inset). The outward current was elicited at
test potentials positive to
20 mV and displayed outward
rectification. It was slowly activating and showed no sign of
inactivation during the depolarizing test pulses.
Ionic basis of the membrane currents.
The granulosa cells incubated on plastic in serum-free medium expressed
a fast-activating/inactivating (transient) inward current or a slowly
activating/inactivating inward (slow) current in combination with the
transient current or a single large inward current that has two
components. These inward currents could result from outflow of
Cl or inflow of Ca2+ among other
possibilities. Experiments were conducted to determine the carrier of
the inward currents in cells that expressed only the large inward
current. In such cells, the reduction of Cl
content of
the external solution resulted in a shift of the reversal potential of
the slowly activating inward current toward the Cl
equilibrium (Fig. 4, A-C,
n = 4 cells). In addition, the slowly activating inward
current was suppressed by the Cl
channel blockers DIDS
(0.5 mM; n = 4 cells; Fig. 4D) and NPPB (20 µM; n = 3 cells, data not shown) in granulosa cells
incubated on plastic, suggesting that the slow inward current is a
Cl
current (caused by outflow of Cl
). A
residual transient current remained after the application of the
Cl
channel blocker (Fig. 4D,
inset). The slowly activating inward current was also
eliminated completely in Ca2+-deficient bath solution in
cells that expressed only one inward current (n = 5 cells; Fig. 5, A-C), and the
general Ca2+ antagonist, cobalt, suppressed the slowly
activating inward current reversibly (n = 4 cells; Fig.
5D). In addition, a combination of the T-type
Ca2+ channel inhibitor (flunarizine) and L-type
Ca2+ channel inhibitor (nifedipine) suppressed the slowly
activating inward current reversibly (n = 4 cells; Fig.
5E). The result shown in Fig. 5 indicates that the slowly
activating inward current, carried by Cl
, was dependent
on extracellular Ca2+. It was hypothesized that the large
inward current is made up of a Ca2+-carried transient
current and a Ca2+-dependent slowly activating
Cl
current. To test this hypothesis, experiments were
conducted with cells that expressed only one inward current in
Ba2+-containing Ca2+-deficient solution; the
transient inward current persisted; however, the larger component of
the inward current was eliminated (Fig. 6, A-C), proving that the
single large inward current is composed of the fast and slowly
activating and inactivating currents. This result also showed that the
fast-activating inward current was carried by Ca2+. (The
persistence of the transient inward current was caused by the
conductance of Ba2+ through specific Ca2+
channels, whereas the component of the inward current, which required
the availability of external Ca2+, was eliminated.) To
substantiate this finding, additional experiments were conducted
with cells that expressed both fast-activating (transient) and slowly
activating inward currents in Ba2+-containing
Ca2+-deficient solution (Fig. 6, D-F). The
substitution of Ba2+ for Ca2+ in the bath
solution resulted in the persistence of the fast-activating transient
inward current and the loss of the slowly activating/inactivating current in a reversible manner, proving that the
fast-activating/inactivating current was carried by Ca2+.
Experiments were also conducted to determine the effect of chelation of
intracellular Ca2+ on the inward current. The addition of 5 mM EGTA to the pipette solution had no appreciable effect on the
activation of the Ca2+-dependent Cl
current
(n = 3, data not shown). This result is consistent with extensive literature showing that the kinetics of Ca2+
buffering by EGTA is slow relative to the kinetics of Ca2+
binding to many Ca2+-activated channels.
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Effect of intact basal lamina on membrane currents and capacitance.
Differentiated granulosa cells incubated on intact basal lamina for
24 h in serum-free medium 199 assumed a spherical shape (with
reduced plasma membrane area), whereas those incubated on plastic in
control dishes became highly flattened with increased total plasma
membrane area (data not shown). The cells were stimulated with a 333-ms
test pulse from a holding potential of 80 mV to depolarizing voltage
steps between
60 and +80 mV. The amplitudes of both inward and
outward currents were measured at the end of the step potentials. The
slow inward current (Ca2+-dependent Cl
current) was suppressed in differentiated granulosa cells incubated on
basal lamina for 24 h (Fig.
8, compare
A and B). The cells were also stimulated with a
10.5-s command pulse from a holding potential of
80 to
40 mV, and
the peak of the inward current was regarded as the amplitude of the
slow (Cl
) inward current (it was measured 250-800 ms
after the onset of the command pulse to exclude the amplitude of the
transient current). The peak amplitude of the slow inward current was
significantly (P < 0.001) greater for cells grown on
plastic than for cells grown on basal lamina (Fig. 8, compare
C and D). When the current amplitude was
normalized to plasma membrane area, the current density of granulosa
cells incubated on basal lamina (n = 43 cells) was
significantly (P < 0.001) smaller than that for cells
grown on plastic (n = 37 cells; Fig.
9, A and B). Basal
lamina had no significant effect on the outward K+ current
under conditions in which it suppressed the slow inward current (Fig.
8, A and B; also Fig. 9, C and
D). In the data shown in Fig. 9, C and
D, the amplitudes of the currents were measured at the end
of the +80-mV step (with a 333-ms stimulus). Membrane capacitance, an
indirect measure of the total plasma membrane area, was significantly
(P < 0.01) smaller for cells cultured on intact basal
lamina (n = 43 cells) than for the control cells incubated on plastic (n = 37 cells; Fig.
9E). The greater the total plasma membrane area, the larger
the membrane capacitance.
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Influence of storage on the effect of intact basal lamina on
membrane capacitance and currents.
Membrane capacitance and currents were recorded in differentiated
granulosa cells incubated in intact basal lamina-containing culture
dishes that had been stored for 12 mo or longer, and the results are
shown in Fig. 10. The membrane
capacitance of cells incubated on intact basal lamina stored for 12 mo
(n = 11 cells) was significantly (P < 0.01) smaller than that in the cells incubated on plastic
(n = 7 cells; Fig. 10A). Also, the density
of the inward Ca2+-dependent Cl current in
control cells (n = 7 cells) was significantly
(P < 0.05) larger than that of cells incubated on the
basal lamina stored for 12 mo (n = 11 cells; Fig.
10B).
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Relationship between membrane capacitance (cell shape) and current.
Because of the apparent consistent relationship between the inward
Ca2+-dependent Cl current and membrane
capacitance in response to basal lamina, correlative decreases in these
two parameters in individual cells were estimated. The amplitudes or
densities of the inward current of granulosa cells incubated on plastic
and basal lamina were plotted vs. their respective membrane
capacitances (Fig. 11). The slopes of
the linear regression were not different from zero (P > 0.05), suggesting that no correlation existed between the amplitudes or densities of the inward current and the membrane capacitance (cell
shape) in granulosa cells incubated on plastic or on basal lamina.
Moreover, the relative change in amplitudes of the inward current
(70-85%) in cells incubated on basal lamina was about threefold
that of the change in membrane capacitance (~22%; see Fig. 9,
A and E). These results suggest that changes in
cell shape alone cannot account for the observed effects of basal
lamina on the inward current.
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Time course of effect of intact basal lamina on membrane currents.
Time course experiments revealed that it required >9 h of incubation
of differentiated granulosa cells on plastic to observe the slowly
activating/inactivating inward current (Fig.
12). The current was not detectable
after 1 h of incubation on plastic; it was expressed modestly
after 6 and 9 h of incubation. The inward current was highly
expressed after 24 h of incubation on plastic. The current was
attenuated in granulosa cells incubated on basal lamina at all time
points tested (Fig. 12). Thus basal lamina appeared to either suppress
the upregulation of the slowly activating/inactivating inward current
or to inhibit the formation of the current-conducting channels.
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Effect of solubilized basal lamina on membrane currents.
In additional experiments, basal lamina was solubilized to enable the
assessment of different amounts of the matrix material on membrane
currents. Differentiated granulosa cells were incubated on coverslips
precoated with different amounts (5 and 15 µg/cm2) of
solubilized basal lamina (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 inward current density (Fig.
13). The effects of solubilized basal lamina were concentration dependent; the current density was reduced 70 and 98%, respectively, by 5 and 15 µg/cm2 of solubilized
basal lamina.
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Effects of cycloheximide and actinomycin D on the slow inward
current.
The effects of the protein synthesis inhibitor cycloheximide and
transcription inhibitor actinomycin D on the slow inward current were
tested. Differentiated granulosa cells were incubated on plastic and
intact basal lamina for 24 h, and membrane currents were recorded.
The cells were stimulated with a 10.5-s command pulse from a holding
potential of 80 to
40 mV, and the peak amplitude of the slow
(Cl
) inward current was measured 250-800 ms after
the onset of the command pulse (to exclude the amplitude of the
transient current). Both cycloheximide (Fig.
14) and
actinomycin D (Fig.
15) inhibited the expression of the slow inward Cl
current in cells
incubated on plastic. Both agents also augmented the inhibitory effects
of basal lamina on the slow inward Cl
current (Figs. 14
and 15). It is noteworthy that cycloheximide or actinomycin did not
inhibit the expression of the transient inward (Ca2+)
current in granulosa cells incubated in the absence or presence of
basal lamina (Fig. 14, B and D, and Fig. 15,
B and D).
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DISCUSSION |
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The results of the present study show that basal lamina of the
preovulatory follicle regulates transmembrane ion transport in
differentiated chicken granulosa cells in a homologous system. Differentiated chicken granulosa cells expressed inward and outward whole cell currents. The Cl channel inhibitors DIDS and
NPPB suppressed the inward current, suggesting that a major component
of this current was carried by Cl
. The activation of this
putative inward Cl
current appears to be dependent on
Ca2+ because the removal of the divalent cation from the
external medium also resulted in the suppression of the inward current.
Results of the present study confirm the earlier report of Mealing et
al. (36) that differentiated chicken granulosa cells express an inward Ca2+-dependent Cl whole
cell current. The present results also confirm the earlier observation
that differentiated chicken granulosa cells express a transient inward
current characteristic of T-type Ca2+ current (16,
36, 49). Furthermore, the outward current observed in the
present study has the characteristics of previously described
delayed-rectifier K+ current in chicken (16, 43,
46) and swine (29) granulosa cells.
In a recent study, a transient outward Cl current could
be activated in differentiated chicken granulosa cells exposed to Ca2+-deficient solution with step potentials between +80
and +120 mV from a holding potential of
80 mV (43). The
transient outward Cl
current was not observed in the
present study, presumably because different step voltage protocols were
used; step potentials between
60 and +80 mV from a holding potential
of
80 mV were employed in the present study.
The observations that membrane capacitance (an indirect measure of
total plasma membrane area) was smaller for granulosa cells incubated
on basal lamina support the results of a previous study in which
morphometric measurements (of cell area, perimeter, and circularity)
were used to demonstrate the effects of basal lamina (intact and
solubilized) on the morphology of granulosa cells (4); the
present and previous studies (4) confirm the observation that basal lamina causes granulosa cells to become rounded. It was
demonstrated that shape per se can regulate cellular functions in
certain instances (15, 26). For example, cell shape
determined whether capillary endothelial cells from human and bovine
origin would undergo apoptosis (15). In addition,
cell shape modulated the control of cell cycle progression in human
capillary endothelial cells (26). This raised the
possibility that the observed effects of basal lamina on inward
Ca2+-dependent Cl current in this study are
consequences of cell shape or that changes in shape or reduction in
membrane area of granulosa cells can account for the observed effects
of basal lamina on the amplitude of the inward current.
Because granulosa cells are exposed to the complex intact basal lamina
as a unit in vivo (but not individual components in isolation), the
effect of basal lamina was examined as a unit. It is presumed that the
action of basal lamina monitored here is the result of the combined
effects of different components of the basal lamina. The nature of the
components of basal lamina that regulated the inward currents and their
mechanisms of action are yet to be determined. Some of the components
are likely to stimulate cellular processes that other components
suppress, although a few 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 the basal lamina of the chicken ovarian
follicle reacted positively to antibodies raised against extracellular matrix proteins (type IV collagen, laminin, entactin, heparan sulfate
proteoglycan, and fibronectin), growth factors (epidermal growth
factor, platelet-derived growth factor, basic and acidic fibroblast
growth factor, insulin-like growth factor, transforming growth
factor-, transforming growth factor-
), cytokines, matrix metalloproteinases, and their tissue inhibitors (8). The
possibility remains that these bioactive molecules acted concomitantly
to regulate the Cl
-carried inward currents in the present studies.
The inhibitory actions of intact basal lamina under the present
experimental conditions were exerted on the inwardly directed Ca2+-dependent Cl current. The ability of the
basal lamina to influence the membrane current was not affected by
solubilization because the total fraction of solubilized basal lamina
(containing all components with molecular mass >3 kDa) also suppressed
the Ca2+-dependent Cl
whole cell current in
granulosa cells in a dose-dependent manner. The concentrations of
solubilized basal lamina used in the present study were shown to
significantly regulate progesterone production in avian granulosa cells
(7).
The observations that cycloheximide and actinomycin D suppressed
the Ca2+ dependent Cl inward current in
control cells suggest that the slow inward (Cl
) currents
in cells incubated on plastic were upregulated by processes that
involved gene transcription and protein synthesis. Because cycloheximide or actinomycin D had little or no effect on the transient
inward current, the actions of the two inhibitors may not be the result
of general inhibition of the cellular processes. The requirement of at
least a 12-h exposure of granulosa cells to the basal lamina
preparations to result in the inhibition of the slow inward
Cl
current suggests that the action of basal lamina is
the result of a long-term (several hours to days) effect of the matrix
material. Although an acute (µs to a few min) effect of basal lamina
on the inward Cl
current should not be ruled out, the
action of basal lamina in the present study is consistent with a
long-term effect. The possibility remains that basal lamina reduced the
number of Cl
channel proteins. Indeed, cycloheximide and
actinomycin D blocked the slow inward Cl
current in the
presence of basal lamina. Although these results do not prove that
basal lamina has a direct effect on the synthesis of channel proteins,
they do not negate the possibility that basal lamina reduced the number
of Cl
channel proteins. The mechanisms that subserve the
actions of basal lamina are yet to be determined.
The results of a recent study showed that both intact and solubilized
basal lamina regulated the shape of differentiated chicken granulosa
cells (4). In additional studies, solubilized basal lamina
(5 and 15 µg/cm2) regulated progesterone production by
differentiated chicken granulosa cells in a dose-dependent fashion
(7). The previous and present results indicate that the
effects of basal lamina on metabolic functions of granulosa cells are
associated with the regulation of the shape and transmembrane ion
transport of these cells. An interesting finding of the present study
is that the storage of basal lamina for 12 or more months at 4°C did
not affect its ability to regulate the inward Cl current.
In relation to this observation is the previous finding that basal
lamina stored for 18 mo or longer regulated the shape of granulosa
cells similar to freshly prepared basal lamina (4).
How do the present observations fit in the known metabolic functions of
differentiated avian granulosa cells? The granulosa cells used in the
present study are differentiated ones isolated from the largest
(F1) preovulatory follicles of hen ovary. These cells are
known to produce large amounts of progesterone (a differentiation marker in avian granulosa cells; see Refs. 7 and 39).
Because solubilized basal lamina suppressed progesterone synthesis in differentiated granulosa cells (7) and because
Cl was shown to be required for progesterone synthesis in
chicken granulosa cells (37), it is possible that the
regulation of transmembrane Cl
transport is a component
of the mechanisms whereby basal lamina suppressed progesterone
production in differentiated granulosa cells (7). It is
noteworthy that the suppression of steroidogenesis in differentiated
granulosa cells exposed to solid basement membrane protein matrixes is
not limited to avian granulosa cells. Notably, Aten et al.
(10) 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 was decreased according to
Aharoni et al. (1). In other studies, when human
granulosa cells retrieved from patients undergoing in vitro
fertilization were cultured in laminin-coated wells, steroidogenesis
was suppressed (22). Similarly, human chorionic
gonadotropin-induced progesterone synthesis was attenuated in
laminin-coated dishes (22). Therefore, reports in the
literature indicate that the incubation of granulosa cells in dishes
precoated with reconstituted or components of basement membranes or
basal lamina results in the reduction of steroid hormone synthesis. The
present data support the hypothesis that Ca2+-dependent
Cl
currents are low in differentiated granulosa cells in
vivo. The data also support the hypothesis that differentiated
granulosa cells that are not in contact with basal lamina or
extracellular matrix in vivo express Ca2+-dependent
Cl
currents, most likely in association with aberrant
metabolic functions.
Because in the present study the Cl-carried inward
current was expressed in granulosa cells that were spread/stretched on plastic, it could be argued that the extensive spreading/stretching was
responsible for the observed increased transmembrane ion transport in
granulosa cells incubated on plastic; perhaps the expression of the
inward Ca2+-dependent Cl
current in granulosa
cells is a stretch-sensitive or a stretch-associated phenomenon. It was
shown that solid elements of the extracellular matrix exert mechanical
stress on cells (28, 51). The nature or type of response
to stress is known to be influenced by the source of stress; for
example, the response of a cell to stress induced by osmotic,
hydrostatic pressures and shear and gravity forces would be expected to
be different from that caused by the extracellular matrix. It follows
that the response of granulosa cells to stress caused by spreading on
plastic would be different from that caused by extracellular matrix,
such as basal lamina (51). Although mechanical stress
imposed by extracellular matrix results in the regulation of cell
function, presumably via tensegrity (tensional integrity) architecture
(18, 28), the nature of the mechanoreceptors that convert
the mechanical stress to intracellular signals is not well defined.
Mechanical stress from the extracellular matrix can be transmitted via
integrins coupled to cytoskeleton but not through nonadhesion receptors
(50, 51). [Integrins are a large family of heterodimeric
transmembrane proteins with different
- and
-subunits that
function as extracellular matrix adhesion receptors
(27).] In addition, stretch-sensitive ion channels
located in the plasma membrane are among the initial sites of action of
mechanical stress (30, 40). Therefore, integrins and ion
channels may serve as transducers of mechanical stress
(mechanotransducers) imposed by different means.
In a tensionally integrated cytoskeleton, the transfer of external mechanical force (mechanotransduction) can cause rearrangements of the cytoskeleton simultaneously at multiple sites in the cell to enable rapid transmission of the mechanical signal, resulting in changes in cellular morphology and function (50). Thus the initial perception of mechanical stress at the cell surface is transmitted to the components of the cytoskeletal system and then to other signaling effectors. Stress-specific Ca2+ channels were activated by stretch in vascular endothelial cells (30). Mechanical force activated stress-sensitive K+ currents in vascular endothelial cells (40), and stretch also activated the Na+/H+ exchanger in cardiac cells (53).
Extracellular matrix proteins have been shown to regulate ion transport in different types of cells. For example, inward whole cell Na+ currents were reduced significantly in rat adult alveolar type II cells cultured on fibronectin-coated coverslips for 48 h (54). Also, rat adult alveolar type II cells cultured on human amniotic basement membrane had smaller short-circuit currents than cells cultured on collagen substrate (19). Furthermore, matrigel (reconstituted basement membrane) modulated K+ current density in fetal rat alveolar type II cells (34).
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 for the study of the effect of basal lamina on membrane
currents in the granulosa cells in a homologous system. Under the
quasiphysiological conditions applied, basal lamina regulated the
membrane ion transport in differentiated granulosa cells. It suppressed
a stretch-sensitive Ca2+-dependent Cl whole
cell current. The observed effects of basal lamina may be a component
of the mechanisms that subserve the reported actions of basement
membranes or other matrix materials on the metabolic functions of
differentiated avian, mammalian, and human granulosa cells.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. R. Shi for comments on the manuscript.
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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 for 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}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 21 May 2001; accepted in final form 24 August 2001.
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