| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Department of Molecular Biology (N.A.G., S.B.), University of Wyoming, Laramie, Wyoming 82071; Department of Chemistry (I.J., T.H.J.), University of Kentucky, Lexington, Kentucky 40506; and Raven Biotechnologies, Inc. (J.P.M.), San Carlos, California 94070
Address all correspondence and requests for reprints to: Dr. Tae H. Ji, Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055. E-mail: tji{at}pop.uky.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
FSH bound the FSH receptor on rat ovarian granulosa cells, and the liganded receptor activated adenylyl cyclase (AC) to produce cAMP but did not mobilize Ca2+. In addition, we observed massive reorganization of the actin cytoskeleton within 3 h of FSH treatment. This involves formation of lamellipodia and filopodia and spreading of multilayer cell aggregates to monolayers. This actin reorganization and cell transformation could also be induced by the AC activator, forskolin, in the absence of FSH. Furthermore, AC inhibitors blocked the FSH-dependent actin reorganization and transformation. On the other hand, phospholipase C inhibitors did not block the FSH-induced changes. Taken together, our observations indicate that the AC/cAMP signal is necessary and sufficient for FSH-dependent granulosa cell differentiation, including massive reorganization of the actin cytoskeleton and changes in the cell morphology and cell-to-cell interactions. There is no evidence that the phospholipase C signal and Ca2+ mobilization are involved in this process.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Studies to determine the effects of FSH on granulosa cell proliferation were not easy, because of the lack of a sustainable and reproducible FSH-responsive culture system. Attempts have been made to develop immortalized granulosa cell lines using oncogenes and oncoviruses (6, 7) and by isolating steroidogenic tumor granulosa cells (8), but these systems often do not express the FSH receptor. Although many cell cultures have been established from ovaries containing follicles of various sizes, the cells are often heterogeneous and respond to FSH differently within the same culture as well as from culture to culture (9, 10). Recently, an undifferentiated granulosa cell line was established from preantral follicles isolated from day-14 Sprague Dawley rats (10). This rat ovarian granulosa (ROG) cell line grows in a defined serum-free medium containing activin A. Upon exposure to FSH, undifferentiated ROG cells differentiate to postmitotic, highly steroidogenic cells with a phenotype similar to mature granulosa cells isolated from a dominant follicle. Once these cells are fully differentiated, they require the continuous presence of FSH and will undergo rapid apoptosis upon FSH withdrawal. Importantly, when oocytes associated with a single layer of cumulus granulosa cells are placed upon a monolayer of ROG cells in culture and stimulated with 30 ng/ml FSH, the ROG cells reorganize around each oocyte to form a multilayer ring structure resembling a follicle (11). This process is quite similar to the steps leading to the in vitro FSH-dependent formation of a follicle and antrum from primary culture cells that were dispersed from small preantral follicles (12).
Taking advantage of this defined ROG system, we set out to determine the early events of hormone signaling during FSH-induced cell differentiation, such as changes in the cell morphology and cell-to-cell interactions. Because structural changes are likely to involve the cytoskeleton, we have examined reorganization of the actin cytoskeleton in ROG cells induced to differentiate by FSH. The actin cytoskeleton is generally involved in cell interactions and tissue development (13). The actin cytoskeleton is also involved in a variety of cellular functions, including intracellular communication, cell polarity, locomotion, establishment and maintenance of morphology, cell-to-cell and cell-to-substratum contacts, and cell division (13). In this article, we report that FSH, but not activin A, induces ROG cell aggregates to first form actin lamellipodia and filopodia, resulting in a cell monolayer. As the cell monolayer is completed and the cell interactions and morphology change, lamellipodia and filopodia diminish, suggesting their involvement in initiating the structural changes but not in maintaining them. In addition, we show evidence that the FSH-dependent adenylyl cyclase (AC)/cAMP signal is necessary and sufficient for these specific and orderly structural changes in ROG cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cell culture
ROG cells were cultured as previously described (10). Briefly,
ROG cells were maintained in suspension in a defined serum-free medium
consisting of F12-DMEM supplemented with activin A (25 ng/ml), insulin
(10 µg/ml), transferrin (5 µg/ml), 
-tocopherol (0.1 µg/ml),
progesterone (10 nM), BSA (0.1%), and aprotinin (25
µg/ml) in the absence of antibiotics. Activin A (25 ng/ml) was
replenished every 24 h. The cells were provided with fresh media
once a week and split every 2 weeks by pooling by centrifugation (1000
rpm for 5 min) and replated at 1:2. Cells not used in an experiment
were allowed to grow until confluent and then either split or used in
the next experiment. ROG cells were not used for experiments past 5
months of continuous culture.
Experimental design
Cells were maintained continuously in T 25 flasks in 5ml of
defined media with activin A until confluency (approximately 3 x
106). For immunofluorescence studies, 200 µl
per condition were removed from the flask and cultured in 8-well glass
chamber slides (Life Science Products, Denver, CO) and allowed
to attach overnight. Before plating, slides were coated with 10 µg/ml
poly-D-lysine and 5 µg/ml fibronectin to facilitate cell
attachment, as per manufacturer’s instruction (Sigma, St.
Louis, MO.). Under these conditions, cell aggregates will attach
loosely to the surface (10). After allowing cells to attach overnight,
the medium was removed and the cells were incubated in the presence or
absence of 1 nM FSH, or activating or inhibitory drugs (as
indicated in figure legends), before being fixed and stained for
F-actin, as described below. Unless otherwise stated, cells are always
cultured in the presence of activin A. To block the cAMP or PLC
pathway, cells were incubated with the appropriate inhibitory drug for
30 min before addition of FSH. Each condition was performed in
triplicate, and 20 cell aggregates were counted per well.
To count proliferation rates in response to FSH, 1 x 105 cells were cultured in 1ml media in 24-well plates and allowed to grow in suspension. Because ROG cells grow as aggregates, they were pipetted up and down vigorously to separate the aggregates into single cells before counting on a hemocytometer.
Fluorescence staining of F-actin
ROG cells grown on 8-well glass chamber slides (Life Science
Products) were fixed and stained for actin. The slides were washed once
in PBS, fixed in 4% paraformaldehyde in PBS at room temperature for 10
min, and permeablized in 0.1% Triton X-100 for 5 min. Cells were then
washed three times in PBS and incubated with PBSBT (1% BSA and 0.05%
Tween-20 in PBS) for 10 min. To stain cells for actin, the slides were
incubated with 0.7 µg/ml TRITC-phalloidin (Sigma)
for 20 min. Cells were then washed three times in PBSBT, at which point
the chambers were removed from the slides, and a coverslip was sealed
in place with clear nail polish.
Confocal microscopy
The fluorescently labeled samples were viewed using a
Leica Microsystems, Inc. (Buffalo, NY) scanning confocal
microscope with a krypton-argon laser. Image series were
acquired and exported to a Macintosh Quadra 700. Projections and
cross-sections were constructed using the NIH image software (written
by Wayne Rasband at NIH and available by anonymous FTP from
zippy.nimh.nih.gov) and pasted into Photoshop 5.0. Cross-sections were
derived from image series stacks encompassing the entire cell layer,
from the coverslip to the top of the cell cluster. A representative
slice from the cell cluster is presented with the coverslip oriented to
the bottom of the panel.
cAMP Assay
cAMP was assayed as previously described (14), with minor
modifications. Cells in suspension culture were collected by
centrifugation, at 200 x g for 5 min at 4 C, and
resuspended in DMEM. Cells in 100 µl DMEM were aliquoted into 1.5-ml
tubes, the tubes were centrifuged to pellet the cells, and the
supernatant was aspirated. The cell pellet was resuspended in DMEM
containing isobutylmethylxanthine (0.1 mg/ml) and incubated at 37 C for
15 min. Increasing amounts of FSH were added, and cells were further
incubated for 45 min at 37 C. The reaction was stopped by pelleting the
cells and aspirating the supernatant. The cells were resuspended in
70% ethanol and lysed by freeze/thawing in liquid nitrogen.
After the cell debris was pelleted by centrifugation at
16,000 x g for 10 min at 4 C. The supernatant was
concentrated under vacuum and resuspended in 10 µl of the cAMP assay
buffer provided by the manufacturer (Amersham Pharmacia Biotech). cAMP concentrations were determined with an
125I-cAMP assay kit (Amersham Pharmacia Biotech, Piscataway, NJ), following the manufacturer’s
instruction and validated for use in our laboratory.
Ca2+ mobilization
Cells were plated on
poly-D-lysine/fibronectin-coated, 8-chambered no. 1
borosilicate coverslip slides (Life Science Products) and allowed to
attach overnight. Cultured cells were incubated in 5 mM
fura-2-acetomethoxyester (fura-2 AM) in DMEM/F-12 without phenol red,
for 1 h at room temperature. Cells were washed twice in DMEM/F-12
without phenol red or fura-2 AM and then incubated in the same media
for at least 30 min to allow for complete deesterification of fura-2 AM
to the Ca2+-sensitive fluorescent indicator,
fura-2. Fura-2-loaded cells were observed continuously, over indicated
times, on a digital imaging system consisting of an inverted
Olympus Corp. microscope, a DeltaRam illuminator, and an
ICCD video camera under computer control of ImageMaster software (PTI,
Princeton, NJ). Fura-2 loaded samples were alternately excited
at 340 nm and 380 nm, and images were stored on a computer for later
analysis. After background subtraction, images were ratioed and
[Ca2+]i calculated
according to published equations (15).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). In contrast,
the cells grown in the presence of activin A, but in the absence of
FSH, have not doubled, even by 96 h. These results indicate that
ROG cells responded to FSH similarly in our hands as in those
previously described (10).
|
As reported by Li et al. (10), in the absence of activin A,
most cells will die within a few days of culture, although FSH slightly
delays cell death but not for long term. In a short period, FSH alone
supported cell spreading to a monolayer on a
poly-D-lysine/fibronectin-coated surface,
although with a morphology different from those treated with both FSH
and activin A (10). Thus, for our study, the actin cytoskeletal
arrangement of ROG cells grown in activin A only was used as a baseline
and control. In ROG cells grown in the presence of activin A only,
actin appeared as cortical actin arranged primarily around the plasma
membrane of each cell (Fig. 2; A, D, and
G). Cortical actin remained, regardless of the incubation time, after
fresh activin A was introduced. When ROG cells were grown in the
presence of both activin A and FSH, lamellipodia (shell-shaped
protrusion) and filopodia (fine spikes) appeared around
the periphery of each cell within 3 h of FSH treatment (Fig. 2B).
To better illustrate these structures, a blowup of the regions of
interest is presented in Fig. 2; A', B', and C'. These lamellipodia and
filopodia were still visible after 6 h (data not shown) and, to a
lesser degree, by 24 h (Fig. 2E). These structures have completely
disappeared by 72 h incubation (Fig. 2H). To determine whether
this actin reorganization requires FSH only or both FSH and activin A,
ROG cells were grown in medium containing FSH but lacking activin A.
Lamellipodia and filopodia appeared again in 3 h after FSH
treatment (Fig. 2C) and have completely disappeared by 72 h (Fig. 2I). These results indicate that formation of lamellipodia and
filopodia requires FSH but not the continuous presence of activin
A.
|
, E and F). By 72 h, the cell clump
has completely transformed into a monolayer (Fig. 2, H and I). These
cell monolayers were more apparent when the cross-section and height of
each cell cluster were examined (Fig. 2, H' and 2I'). In cross-section,
the individual cell cluster appeared as a monolayer one-cell-layer
thick, in cultures exposed to FSH (Fig. 2, H' and 2I'), compared with
the cross-section of the multilayer cells that were not treated with
FSH (Fig. 2G').
In addition to these differences between the cells treated with FSH and
those without FSH treatment, we observed distinct morphological and
cytoskeletal differences between the cells treated with FSH in the
presence and absence of activin A. As can be seen when comparing
Fig. 2H with Fig. 2I, it seems that cells grown in the presence of both
FSH and activin A are smaller and more rounded than those cultured in
the presence of FSH without activin A. In the latter cells, the actin
cytoskeleton also appeared slightly more randomly arranged. Therefore,
activin A may also impact the actin cytoskeleton but does so
differently from the FSH-induced lamellipodia and filopodia formation.
Because FSH treatment results in the formation of lamellipodia and
filopodia both in the presence and absence of activin A, and the cell
morphology of FSH and activin A-treated cells resembles that of
hyperstimulated primary cultures of granulosa cells (10), all further
experiments were conducted in the continuous presence of activin A.
FSH induces lamellipodia and filopodia formation via the AC
signal
The results in Fig. 2 indicate that ROG cells responded to FSH,
forming lamellipodia and filopodia within 3 h of FSH treatment
(Fig. 2, B and C) and spread to form a near monolayer by 24 h
(Fig. 2, E and F). However, the signal mechanism is unclear. To
determine whether activation of AC in the absence of FSH will cause the
same changes, ROG cells were treated with an AC activator, 10
µM forskolin (16), or an AC inhibitor, SQ 22536 or
MDL-12536 (17, 18) without FSH. Similar to the cells treated with FSH,
forskolin-treated cells developed lamellipodia and filopodia within
3 h (Fig. 3, B and B') and spread to
form monolayers by 24 h (Fig. 3E). Again, to highlight the regions
of interest, a blowup of the lamellipodia and filopodia can be viewed
in Fig. 3; A', B', and C'. To determine the percentage of the cells
with lamellipodia and filopodia, cell aggregates that had spread were
counted among all aggregates, under 40x microscopic view, after
24 h incubation. Discrete cell monolayers were seen in the entire
population of the cells examined, indicating forskolin induced actin
reorganization in 100% of the cells. To control for any side effects
of the dimethylsulfoxide used to dissolve forskolin, cells were
incubated in medium containing the same concentration of
dimethylsulfoxide used in the for-skolin experiments. Cells treated
with dimethylsulfoxide looked similar to untreated cells (data not
shown). To test unknown side effects of forskolin other than activation
of AC, ROG cells were incubated with 10 µM 1,9-dideoxy-
forskolin, a naturally occurring analog of forskolin that does not
activate AC. Again cells treated with 10 µM
1,9-dideoxy-forskolin did not form lamellipodia and filopodia or spread
(data not shown). To further test whether the AC signal is responsible
for actin rearrangements leading to lamellipodia and filopodia
formation, ROG cells were exposed to either of two different AC
inhibitors, SQ 22536 or MDL-12,330A, before FSH stimulation. After
exposure of ROG cells to 1 mM SQ 22536 for 30 min, FSH
failed to induce lamellipodia and filopodia formation by 3 h (Fig. 3, C and C'). Similarly, only 10–15% of the cell clusters had spread
by 24 h (Fig. 3F). These numbers increased to 85–90% spreading
when cells were exposed to SQ 22536 diluted 100-fold to 0.01
mM, indicating drug specificity. Similar results were
observed when the cells were exposed to 25 µM MDL-12,330A
for 30 min before FSH stimulation. In this case, 20–25% of the ROG
cell aggregates spread (data not shown). As with SQ 22536, 90% of the
cell aggregates spread when cells were exposed to MDL-12,330A diluted
to 0.25 µM.
|
). This response can be
completely blocked by the AC-inhibiting drug, SQ 22536. ROG cells were
preincubated with 1 mM SQ 22536 for 30 min, before addition
of FSH, and assayed for cAMP accumulation. As indicated in Fig. 4A, FSH
is unable to cause accumulation of cAMP when the AC pathway is blocked.
To ensure that cells treated with an AC-inhibiting drug remain viable
and able to respond to cAMP, cells were preincubated for 30 min in SQ
22536 with a cAMP analog, 10 µM 8-bromo-cAMP, for 24
h (Fig. 4B). These cells display a few lamellipodia and filopodia and a
phenotype similar to cells treated with FSH, with flattened cells on
the periphery of aggregates and few rounded cells in the center.
|
), similar to those seen in FSH-induced
cells (Fig. 5A). These results could be repeated by incubating the
cells with 20 µM dibutyryl-cAMP over the same time period
(data not shown). Because cAMP primarily activates PKA, we decided to
see whether activation of PKA is also involved in lamellipodia and
filopodia formation. For this purpose, we treated ROG cells with either
of two PKA inhibitors, H-89 and KT5720 (21, 22, 23), for 30 min before
either FSH addition (Fig. 5B) or 8-bromo-cAMP (Fig. 5D). Whether cells
were treated with FSH or 8-bromo cAMP, spreading was almost completely
abolished in the presence of 10 µM H-89 for 24 h.
Another PKA inhibitor, KT5720, was similarly effective in blocking
actin rearrangements induced by either FSH or 8-bromo-cAMP (data not
shown). These results clearly indicate that FSH is capable of inducing
cAMP production, which is necessary and sufficient for lamellipodia and
filopodia formation and cell spreading to form monolayers. In addition,
our data show that PKA is involved in these changes.
|
, 10 µM U-73122 did not
impact FSH-dependent formation of lamellipodia and filopodia. In
addition, 85%–90% of the cell aggregates spread to form a monolayer
by 24 h, in response to FSH (Fig. 6B). Similar results were
observed with 15 µM ET-18-OCH3, another PLC inhibitor
(data not shown). In this case, 80%–85% of the cell aggregates
spread to form a monolayer by 24 h. Taken together, there is no
evidence that the PLC signal is involved in FSH-induced lamellipodia
and filopodia formation.
|
, A–C).
The result was the same, even when the cells were treated with 3.3
nM FSH (data not shown), at which cAMP is maximally induced
(Fig. 4).
|
). The cells had
completely recovered by 90 sec, with Ca2+
concentrations returning to basal levels of 75 nM (Fig. 7F). To ensure the ATP-induced Ca2+ increases
were a result of PLC activation and, at the same time, prove that
U-73122 is able to block the PLC pathway in ROG cells, cells were
treated with 10 µM U-73122 before ATP addition. The cells
were assayed continuously over the same time course previously
determined necessary for Ca2+ increase and the
subsequent cell recovery. When ROG cells were treated with U-73122, ATP
was unable to mobilize Ca2+ (Fig. 7, G–I). The
result was the same when 15 µM ET-18-OCH3, a different
PLC inhibitor, was used (data not shown). These results indicate that
the PLC pathway leading to Ca2+ mobilization is
functional in ROG cells but is not connected to the FSH receptor. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
FSH is known to activate AC and induce cAMP synthesis in granulosa and Sertoli cells (24, 46, 47), as well as in transfected mammalian cells (25). cAMP activates PKA, which, in turn, phosphorylates structural proteins, other enzymes, and transcriptional factors leading to gene regulation (48). The effects of FSH on the PLC pathway and the associated second messengers, diacylglycerol and inositol phosphates, are less clear and controversial. For example, FSH has been suggested to play no significant role in phosphatidylinositol turnover in Sertoli cells (49) or inhibit inositol phosphate accumulation via the AC pathway (50). In contrast to these reports, it has been reported that FSH stimulates the phosphatidyl inositol turnover, thus activating both protein kinase C and intracellular Ca2+ mobilization in cumulus cell-enclosed oocytes. In this case, FSH induced oocyte meiotic resumption via the PLC pathway (51). Rat FSH receptors transiently or permanently expressed in human embryonic kidney 293 cells are capable of activating the PLC pathway, and they stimulate inositol phosphate accumulation in response to FSH (26, 27). It is important to note, however, that overexpression of seven transmembrane receptors may result in inappropriate coupling to signal pathways (48, 52). Thus, although the FSH receptor is capable of activating both the AC and PLC signal pathways in a transfection system like human embryonic kidney 293 cells, the dual signal generation in vivo or in cells in which the receptor is naturally expressed, like granulosa and Sertoli cells, remains unclear. Our results indicate that the AC signal, in particular cAMP, is responsible for lamellipodia and filopodia formation and the subsequent changes. We find no evidence that the PLC signal, in particular Ca2+, is involved, although the PLC signal pathway leading to Ca2+ mobilization is functional in ROG cells. In fact, we find no evidence that the FSH receptor is activated by FSH in this cell line. Although the FSH receptor was reportedly linked to extrinsic Ca2+ uptake by swine granulosa cells (53, 54), it should be noted that the mechanism of Ca2+ uptake from external sources is entirely different from Ca2+ mobilization from internal stores. External uptake is not dependent on IP3 generation via PLC activation as is Ca2+ mobilization from the ER. In fact, CA2+ was excluded from the medium to ensure that only intracellular Ca2+ mobilization caused by PLC activation was detected in our assay.
So far, our data suggest that the FSH/FSH receptor/AC/cAMP pathway leads to filopodia and lamellipodia formation. It would be interesting to determine the factors downstream of PKA that are activated to induce the formation of these podia. It could be speculated that the small-molecular-weight G proteins, Rac and Cdc42, are involved here. Assembly and reorganization of the actin cytoskeleton in other types of cells are regulated by several Rho subfamily members (Rho, Rac, and Cdc42) of the Ras GTPase family (13, 55). Rac activation leads to formation of the actin filament network at the cell periphery, to produce lamellipodia, whereas Cdc42 induces the actin-rich surface spikes called filopodia. As these Rho family GTPases cross-talk (13), lamellipodia and filopodia are often formed simultaneously, as seen in ROG cells. These actin structures are also associated with integrin-based cell adhesion complexes and, therefore, involved in cell interactions. This may explain why assembly of lamellipodia and filopodia in ROG cells is associated with the transformation of the cell aggregates to monolayers.
The signal pathways of different receptors leading to individual Rho GTPases are poorly defined. To our knowledge, the signal pathway from the FSH receptor/AC to lamellipodia and filopodia formation in ROG cells is novel. Our observations also raise the potentially significant possibility that the AC signal passes through Rac and Cdc42 to induce the formation of lamellipodia and filopodia. Recently, it has been described that FSH activates p38 mitogen-activated protein kinase via AC/cAMP in immature ROG cells (56). It will be interesting to determine whether the pathway from the FSH receptor/AC/cAMP to the Rho GTPase family overlaps with the pathway to p38 mitogen-activated protein kinase.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received January 20, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-subunit expression by follicle-stimulating hormone
[in process citation]. Biol Reprod 58:219–225This article has been cited by other articles:
 |
|
 |
|
  C. Ko, N. A. Grieshaber, I. Ji, and T. H. Ji Follicle-Stimulating Hormone Suppresses Cytosolic 3,5,3'-Triiodothyronine-Binding Protein Messenger Ribonucleic Acid Expression in Rat Granulosa Cells Endocrinology, June 1, 2003; 144(6): 2360 - 2367. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. A. Grieshaber, C. Ko, S. S. Grieshaber, I. Ji, and T. H. Ji Follicle-Stimulating Hormone-Responsive Cytoskeletal Genes in Rat Granulosa Cells: Class I {beta}-Tubulin, Tropomyosin-4, and Kinesin Heavy Chain Endocrinology, January 1, 2003; 144(1): 29 - 39. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Sasson and A. Amsterdam Stimulation of Apoptosis in Human Granulosa Cells from in Vitro Fertilization Patients and Its Prevention by Dexamethasone: Involvement of Cell Contact and Bcl-2 Expression J. Clin. Endocrinol. Metab., July 1, 2002; 87(7): 3441 - 3451. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. T. Budnik and A. K. Mukhopadhyay Lysophosphatidic Acid Antagonizes the Morphoregulatory Effects of the Luteinizing Hormone on Luteal Cells: Possible Role of Small Rho-G-Proteins Biol Reprod, July 1, 2001; 65(1): 180 - 187. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. A. McGowan, M. Madesh, Y. Zhu, L. Wang, M. Russo, L. Deelman, R. Henning, S. Joseph, G. Hajnoczky, and K. Sharma TGF-beta -induced Ca2+ influx involves the type III IP3 receptor and regulates actin cytoskeleton Am J Physiol Renal Physiol, May 1, 2002; 282(5): F910 - F920. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
