Identification of an Ovarian Voltage-Activated Na+-Channel Type: Hints to Involvement in Luteolysis
Andreas Bulling,
Frank D. Berg,
Ulrike Berg,
Diane M. Duffy,
Richard L. Stouffer,
Sergio R. Ojeda,
Manfred Gratzl and
Artur Mayerhofer
Anatomisches Institut der Technischen Universität
München (A.B., M.G., A.M.) D-80802 München,
Germany
Frauenklinik der Ludwig Maximilians-Universität
(F.D.B., U.B.) D-80333 München, Germany
Division of Reproductive Sciences (D.M.D., R.L.S.) and
Division of Neuroscience (S.R.O.) Oregon Regional Primate Research
Center Oregon Health Sciences University Beaverton, Oregon
97006
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ABSTRACT
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An endocrine type of voltage-activated sodium
channel (eNaCh) was identified in the human ovary and human luteinized
granulosa cells (GC). Whole-cell patch-clamp studies showed that the
eNaCh in GC is functional and tetrodotoxin (TTX) sensitive. The
luteotrophic hormone human CG (hCG) was found to decrease the peak
amplitude of the sodium current within seconds. Treatment with hCG for
2448 h suppressed not only eNaCh mRNA levels, but also mean
Na+ peak currents and resting membrane
potentials. An unexpected role for eNaChs in regulating cell
morphology and function was indicated after pharmacological modulation
of presumed eNaCh steady-state activity in GC cultures for 2448 h
using TTX (NaCh blocker) and veratridine (NaCh activator). TTX
preserved a highly differentiated cellular phenotype. Veratridine not
only increased the number of secondary lysosomes but also led to a
significantly reduced progesterone production. Importantly, endocrine
cells of the nonhuman primate corpus luteum (CL), which represent
in vivo counterparts of luteinized GC, also contain eNaCh
mRNA. Although the mechanism of channel activity under physiological
conditions is not clear, it may include persistent
Na+ currents. As observed in GC in culture,
abundant secondary lysosomes were particularly evident in the
regressing CL, suggesting a functional link between eNaCh activity and
this form of cellular regression in vivo. Our results
identify eNaCh in ovarian endocrine cells and demonstrate that their
expression is under the inhibitory control of hCG. Activation of eNaChs
in luteal cells, due to loss of gonadotropin support, may initiate a
cascade of events leading to decreased CL function, a process that
involves lysosomal activation and autophagy. These results imply that
ovarian eNaChs are involved in the physiological demise of the
temporary endocrine organ CL in the primate ovary during the menstrual
cycle. Because commonly used drugs, including phenytoin, target NaChs,
these results may be of clinical relevance.
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INTRODUCTION
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The corpus luteum (CL) in primates forms after ovulation and
represents a temporary endocrine organ within the ovary (1 2 ). The
main function of the CL during its functional life span is the
production of the steroid progesterone, which is fundamental for
preparation and maintenance of pregnancy. Both the CL structure and
progesterone are hormonally controlled by the pituitary hormone LH or,
during pregnancy, by a closely related placental hormone (CG). The
presence or absence of these luteotrophic hormonal signals determines
growth and survival of the highly differentiated CL or its demise. In
nonpregnant women this process of formation, growth, and regression of
the CL occurs during each ovarian cycle. It is not clear what initiates
the regression of the CL at the end of each menstrual cycle, in
particular if the altered, lower frequency of pituitary LH pulses is of
importance (2 3 ). However, in a nonhuman primate species, cynomolgus
monkeys (Macaca fascicularis), luteal regression and reduced
progesterone production are caused by a reduction in the responsiveness
of the aging CL to LH or hCG (4 ). The observed shutting down of
progesterone production is often referred to as functional luteolysis.
The functional demise of the CL is followed by a longer lasting
process, structural luteolysis, a term describing the remodeling of the
CL into a scar, during which the luteal cells are replaced by
connective tissue.
Tight control of the luteolytic process during the menstrual cycle and
after pregnancy is of pivotal importance for ovarian and reproductive
physiology. However, the mechanisms responsible for the initiation of
luteolysis at the cellular level are not well understood. In many
species, apoptosis of luteal cells has been implicated in this process.
In the rat, for example, apoptosis-associated genes become expressed in
luteal cells (5 6 ), and the typical programmed cell death signs
including DNA fragmentation are well documented (7 8 9 ). In nonhuman
primates and humans, the situation is less clear. Although cultured
human granulosa cells express both the Fas antigen (10 ) and the
apoptosis-inducing protooncogene product BAX, no evidence for apoptosis
was found in luteal cells in human CL examined for DNA fragmentation
using the terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP
nick end labeling (TUNEL) method (11 12 ). In another report, some
scattered TUNEL-positive cells were reported in the degenerating human
CL (13 ). Likewise, apoptotic cells were found in the regressing CL
(14 ), which, however, were mainly vascular cells. It is possible that
these cells may account for DNA fragmentation reported by other
investigators (13 15 16 ) in degenerating human CL. Endocrine cells of
the human CL may be protected by the product of the protooncogene
bcl-2, an inhibitor of apoptosis, which was demonstrated in luteal
cells throughout the luteal phase (17 ). It is thus unclear whether
apoptosis is a major contributor to either the functional or the
structural luteolysis in humans. In the nonhuman primate ovary of the
marmoset (18 19 20 ), apoptosis of luteal cells was detected, but another
morphologically distinct form of cell regression became apparent. These
luteal cells were characterized by the formation of cytoplasmic
vacuoles due to cellular atrophy and phagocytosis of cytoplasmic
debris. This process in primates, beyond its phenomenological
description at the light microscopic and ultrastructural level (19 20 ), was not further examined. Equally unknown is the relationship that
may exist between this form of cellular regression and the drastic
increase in the number of lysosomes and lysosomal activity described,
in particular, in the regressing CL of several species (21 22 )
including the human (20 23 ).
In a preliminary study (24 ), we reported the presence of functional
voltage-activated potassium and sodium channels (NaCh) in human
luteinized granulosa cells (GC). The type present in freshly isolated
and cultured GC was found to be homologous to the one originally
described in neuroendocrine cells of the adrenal and thyroid C cells
(25 ). We named it endocrine (e) NaCh, rather than neuroendocrine,
because of its presence in steroidogenic cells. In an attempt to
characterize ovarian eNaCh and to elucidate its function, we report
here evidence that the eNaCh type is also expressed in the human ovary
and in the CL of nonhuman primates. Our results indicate hormonal
down-regulation of these ion channels and show the consequences of
eNaCh activation on the secretory function and lysosomal activation of
progesterone-producing cells.
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RESULTS
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RT-PCR Identification of Ovarian eNaCh
We amplified cDNAs of 222 bp from human ovary and human GC
(cultured for 2, 3, and 9 days) (Fig. 1
).
After sequencing of the PCR products, we found that they corresponded
to the
-subunit of an endocrine (e) voltage-activated NaCh. The form
in GC (two clones from culture day 2; two clones from culture day 3;
one clone from day 9; one clone from ovary) was 100% identical to a
previously identified neuroendocrine NaCh (25 ). When Northern blot
analyses were performed with RNA extracted from human GC (Fig. 2
), an eNaCh transcript of approximately
7.5 kb was detected in untreated cells cultured for short or longer
periods of time (days in vitro, DIV, 110).

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Figure 1. Ethidium Bromide-Stained Agarose Gel Showing
cDNAs Obtained by RT-PCR
They correspond to eNaCh as confirmed by sequence analysis and were
identified in the human ovary (A) and cultured human GC (B, 2 DIV).
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Figure 2. Detection of eNaCh mRNA in Untreated and
hCG-Treated Human GC, Using Northern Blotting
An eNaCh transcript at approximately 7.5 kb is found in human GC
cultured for 24 h in the absence (Co) or presence of 10 IU/ml hCG.
The same blots were subsequently probed for ß-actin (1.3 kb),
which was used for normalization of the results.
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Electrophysiological Characterization of GC eNaCh
The mean resting membrane potential of human GC measured directly
after establishing the whole-cell configuration was -32.7 ± 9.1
mV (n = 6 cells; 4, 7, and 8 DIV). Using the standard internal
solution, the pulse protocol for activation gave rise to transient
inward and outward currents (Fig. 3A
) as
described previously (24 ). With the solution containing CsCl/
tetraethylammoniumchloride (TEA)-Cl, outward currents were blocked
(Fig. 3B
), indicating the presence of voltage-activated
K+ channels. The remaining transient inward
currents were sensitive to tetrodotoxin (TTX, Fig. 4A
), indicating the presence of voltage-
activated Na+ channels. Increasing
concentrations of TTX allowed determination of the half-maximal
blocking concentration (IC50) of 6.8
nM (Fig. 4B
). A Hill slope of 0.67 was determined,
differing from the expected value of 1. Maximum inward currents were
obtained at voltage steps from -120 mV to a test potential of -15 mV,
and the resulting Na+ current densities ranged
from -2 to -30 picoamperes (pA)/picofarads (pF). Assuming a general
value for NaCh single channel conductance of 15 picoSiemens (pS) (26 ),
the maximum density was estimated to be 0.3 channels per
µm2 cell membrane.

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Figure 3. Results from Patch-Clamp Recordings of Human GC
A, Transient inward and outward currents are elicited by stepwise
depolarization (+15 mV per step) from a holding potential of -120 mV
to various test potentials between -90 and +45 mV. Inward currents
(arrow) are not resolvable at the time scale shown. B,
Patch pipettes filled with CsCl/TEA-Cl blocked outward currents,
leaving only TTX-sensitive inward currents with maximum amplitudes of
up to 1.5 nA. Increase of test potentials +5 mV per step.
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Figure 4. TTX Sensitivity of eNaCh in GC
A, Effect of increasing concentrations of TTX on transient inward
currents of one cell. From bottom to top
trace: 0, 0.1, 1, 10, 100, and 1000 nM TTX. B, Results from
eight different cells reveal a value for IC50 of 6.8
nM and a Hill slope of 0.67 for inhibition by TTX
(circles are single values or means ±
SD).
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Although the measured current traces resembled those underlying
excitation processes, no action potentials could be triggered when
depolarizing currents were injected in current-clamp mode. Results from
Boltzmann fits (Fig. 5
) of steady-state
inactivation revealed a mean V50 value of
-70.4 ± 8.8 mV (n = 13; 311 DIV) that was independent of
current density or culture time. In contrast, V50
values of activation showed a significant shift to more negative
potentials (P < 0.01, F Test, n = 30) from
approximately -10 to -20 mV over a culture period of 10 days (not
shown). For individual cells, the proportion of open eNaCh allowing
persisting Na+ fluxes, based on the model of
Hodgkin and Huxley (27 28 ) (window current), is between 0.3% and
4.0%.

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Figure 5. Example of Na+ Current Activation ()
and Steady-State Inactivation ( ) in a GC
In this specific cell (3 DIV), superposition of both curves would
result in a window current (dashed line) of up to 2% of
maximal Na+ current at a potential of -25 mV.
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Regulation of eNaCh by hCG
hCG was able to regulate both eNaCh mRNA level and eNaCh function.
When the luteotrophic hormone hCG (10 IU/ml) was added to cultures of
human GC for 2448 h, eNaCh mRNA levels were suppressed (Fig. 2
).
Results in three independent experiments showed reduction beyond
detection level of eNaCh in one experiment (24 h treatment with hCG),
reduction to 36% of control levels in another experiment (24 h), and
reduction to 68% when cells were treated for 48 h.
Incubation of human GC with hCG for 48 h reduced the resting
membrane potential (P < 0.05; n = 30 control
cells, 14 treated cells; 13 DIV) from -26.8 mV to -19.3 mV (Fig. 6
). Moreover, after this period the
specific Na+ currents were significantly reduced
(Fig. 6
; control group 30 cells: -9.50 pA/pF; hCG group: -3.46 pA/pF;
n = 21 cells; P < 0.01). In addition, hCG was
found to have an acute and profound effect on the peak amplitude of
voltage-activated Na+ current. In 10 of 10
experiments (using 5 cells) we observed that within seconds after hCG
application, the peak amplitude of the Na+
current was reversibly lowered (Fig. 7
).

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Figure 6. Regulation of Na+ Current and Membrane
Potential by Chronic hCG Treatment
Incubation of human GC with hCG (10 IU/ml, 48 h, from DIV 13),
reduced specific Na+ currents, and membrane resting
potentials when compared with untreated control cells. In panel A, the
mean currents (marked by horizontal lines) were -9.50
pA/pF and -3.46 pA/pF; P < 0.01 (t
test); n = 30 control cells/21 treated cells. In panel B, the mean
potentials were -26.8 mV and -19.3 mV; P < 0.05
(t test); n = 30 control cells/14 treated cells.
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Figure 7. Acute Effect of hCG on eNaCh Activity
Application of hCG (10 IU/ml) to GC (3 DIV) reversibly reduced the peak
amplitude of voltage-activated Na+ currents. Similar
results were seen in 10 of 10 experiments with 5 different cells. The
inset shows current traces obtained 10 sec before, at
application of, and 10, 12, 14, 16, and 18 sec after application of
hCG.
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Consequences of eNaCh Activation and Blockage in GC: Progesterone
Production and Lysosomes
Activation of eNaCh by 50 µM veratridine for 2448
h decreased media progesterone levels by half (P <
0.05), whereas TTX showed no statistically significant effect (Fig. 8
). Blockage of eNaCh by TTX for 2448 h
preserved a highly differentiated cellular phenotype (Fig. 9
). Electron microscopy showed GC with
abundant intact mitochondria, smooth and rough endoplasmic reticulum,
and lipid droplets. In contrast, activation of eNaCh by veratridine
induced abundant secondary lysosomes, characterized by their
heterogeneous content. Most of them appeared to be autophagosomes
containing remnants of cytoplasmic organelles. The increase in
lysosomes resulting from veratridine treatment of GC was also confirmed
by the visualization of accumulation of a lysosomal-specific dye in GC
(Fig. 10
). There was no evidence for
apoptosis; in particular, nuclei of veratridine-treated cells were not
altered. Concomitant incubation with TTX was able to prevent the
veratridine effects (Fig. 9F
). None of the described changes were seen
when human carcinoma cells (A431) lacking NaCh were incubated with
veratridine or TTX (not shown).

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Figure 8. Consequences of eNaCh Activation and Blockage on
Progesterone Production
Incubation of GC with veratridine (50 µM) for 24 h
or 48 h in GC (culture days 1 2 or 13, respectively) decreased
progesterone production by half compared with untreated control cells
(P < 0.05, marked by asterisks),
whereas TTX (5 µM) showed no statistically significant
effect. Bars represent means ± SEM.
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Figure 9. Consequences of eNaCh Activation and Blockage on
Cell Morphology
A, Ultrastructural features of a GC cultured in basal medium. Note
mitochondria (double arrow) and lipid droplets
(asterisks), as well as some lysosomes
(arrows). Bar, 2.5 µm. B,
Ultrastructure of a GC cultured for 24 h in basal medium and then
treated with TTX (5 µM) for 24 h: note the
abundantly present mitochondria (double arrow), few
lysosomes (arrows) and lipid droplets
(asterisks). Bar, 2 µm. C, Veratridine
(VERA, 50 µM) treatment (24 h) led to the appearance of
abundant large secondary lysosomes (arrows). Most of
them appeared to be autophagosomes. Note that the nucleus is not
altered. Few mitochondria (double arrow) and lipid
droplets (asterisks) are seen. Bar, 2.5
µm. D and E, Examples of the morphology of the secondary lysosomes
found in veratridine-treated GC. Bars, 0.8 µm. F, TTX
treatment blocked the effects of veratridine. Note presence of abundant
mitochondria and absence of lysosomes. Bar, 1.5 µm.
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Figure 10. Increased Lysosomes in GC after Veratridine
Treatment (VERA, 50 µM) in a Lysosomal Dye Experiment
An increase in lysosomes (red) over the numbers seen in
untreated cells (Co; A) was observed in veratridine-treated GC for
24 h (B) as visualized by a lysosomal dye. Bars, 50
µm.
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Expression of eNaCh in the Primate CL
Using RT-PCR followed by sequence analysis, we found that the form
of the eNaCh was also present in the rhesus monkey ovary and CL on days
3 (two clones), 10, and 14 (one clone each) corresponding to the early,
mid, and late life phase of this organ (Fig. 11
). Human and monkey eNaCh sequences
did not differ. The monkey sequence was submitted to Genbank (accession
no. AF164965).

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Figure 11. Presence of eNaCh mRNA in the Monkey CL and Ovary
Ethidium bromide visualization of RT-PCR-derived cDNAs corresponding to
eNaCh and cyclophillin (Cycloph.) are shown. Identity of cDNAs was
confirmed by sequencing. The eNaCh mRNA is present in the early (E),
mid (M), and late (L) phase of the life cycle of the CL, as well as in
a monkey ovary (MK-Ov).
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Electron Microscopy of Luteal Cells
Electron microscopic examination of the CL from nonhuman primates
revealed that on days 7 and 11, i.e. when the CL is
functional, luteal cells contain abundant primary lysosomes (Fig. 12
), characterized by their homogenous
content, and also a few secondary lysosomes. Numerous secondary
lysosomes prevailed later in the regressing CL (days 15, 16, and 18),
indicating that they can be induced not only in GC in vitro,
but are also present in normal cells of the regressing CL near the end
of the menstrual phase. Thus the observations in GC are mirrored by
results obtained in vivo.

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Figure 12. Electron Microscopic Features of Monkey CL
A, Electron microscopical examination of a monkey luteal cell of day 7
of the luteal phase. Note primary and secondary lysosomes
(arrows). Bar, 1.3 µm. B, A
luteal cell from day 11 of the luteal phase showed primary and
secondary lysosomes (arrows). Bar, 1 µm. C,
Secondary lysosomes were present in a regressing luteal cell CL (day 18
of the luteal phase). Bar, 1.3 µm.
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DISCUSSION
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In the present study we report that the gene for a member of the
voltage-activated, TTX-sensitive family of Na+
channels is expressed in steroid-producing endocrine cells of the
primate ovary. Northern blotting and RT-PCR experiments showed the
presence of eNaCh in vitro in human luteinized GC and in the
corresponding tissue in vivo, in the rhesus monkey CL. This
channel type was previously described in humans to be expressed in
neuroendocrine cells (C cell carcinoma), as well as in bovine adrenal
and thyroid, but not in pituitary or brain (25 ). Rat homologs were
subsequently described in peripheral neurons of the dorsal root
ganglion (29 30 ). This widespread distribution indicates that this
form of NaCh is not as specific for neuroendocrine cell types, as
previously suggested. Although the monkey ovary contains neuron-like
cells (31 32 ), which may also express NaCh (our unpublished
observation), these cells are found exclusively in ovarian stroma and
not within the CL. This fact and the current observation, that the
counterparts of luteal cells, isolated human GC, express the eNaCh
gene, clearly identify steroidogenic cells as the sites of expression
of eNaCh and exclude contamination by other cells.
The mRNA encoding the eNaCh was present throughout the life span of the
monkey CL, during development (day 3), function (day 10), and
regression (day 14) in the luteal phase of the menstrual cycle. A
preliminary semiquantitative RT-PCR study of small CL samples
(our unpublished observation) suggested that overall mRNA levels
of the eNaCh increase toward the end of the life span of the CL. The CL
is a heterogeneous organ, composed of different cell types (endocrine,
vascular, immune, and connective type of cells), and their composition
changes dramatically during the life of the CL (2 33 ). Examination of
only a part of the CL is therefore not truly reflecting the complex
events inside this endocrine organ and thus, we did not follow up on
this preliminary result. Rather we attempted to study eNaCh function
and regulation in a pure cell culture system of luteinized GC. In this
system, we found, unexpectedly, that the luteotrophic hormone hCG
markedly decreased eNaCh mRNA levels, probably indicating a negative
hormonal regulation of eNaCh transcription. This was accompanied by
altered resting membrane potential and Na+
currents. Moreover, hCG directly reduced Na+ peak
currents within seconds. These effects of a gonadotropin are important
in view of CL function in vivo. In the primate, progesterone
production of the aging CL drops and LH secretion pattern changes (2 ).
Alterations of LH frequency and amplitude, however, appear not to
account for the regression of the function of this organ. Luteolysis
can, as recently shown in an elegant study, be overcome simply by
additional LH/hCG infusions (4 ). Thus the decline in CL function can be
prevented by a stronger gonadotropin support. This result proves that
it is the responsiveness of this temporary endocrine organ to LH/hCG
that becomes dramatically reduced at the end of its life span (4 ). The
precise reasons for this reduced responsiveness are not fully clear.
One likely reason is a drop in vascular support of this tissue.
Consequently, an individual luteal cell in the CL experiences a
reduction in available or bioactive LH/hCG, i.e. a situation
simulated in human GC cultured without hCG.
In general, the factors regulating the expression of the different NaCh
genes are largely unknown (29 ) and, to our knowledge, a repressive
function on NaCh gene expression has not been reported in any other
cells. In contrast, that a closely related NaCh, the one described in
astrocytes, can be positively regulated by factors originating from
neurons, is well documented (34 ). But the nature of these, as yet
undefined, factors present in conditioned medium from cultured neurons
is unknown. In GC, it remains to be clarified whether hCG itself or
hCG-induced gene products are the true inhibitors of NaCh gene
transcription. For example, in addition to secretion of progesterone,
hCG stimulates release of relaxin and oxytocin (35 ). That hCG has an
acute and direct effect on the function of existing eNaCh, however, was
clearly demonstrated by its ability to reduce the peak amplitude of
Na+ currents in repeated pulse experiments within
seconds. It is important to mention that this immediate effect of hCG
is also of inhibitory nature. It is conceivable that hCG may act via
cAMP and subsequent phosphorylation of the channel protein, because
such a mechanism has been shown to exist in neurons that express
another type of a NaCh (36 ). Thus, our data indicate that eNaChs
are negatively regulated directly and/or indirectly by the luteotrophic
hormone LH/hCG, which is crucial for the functional competence of the
CL. Our results support the hypothesis that LH/hCG suppresses both
eNaCh expression and function and thus is able to prevent deleterious
downstream events that may result from the activation of this ion
channel (see below).
Voltage-activated sodium channels constitute a family of related ion
channels, which are expressed mainly, but not exclusively, in excitable
cells, such as neurons, smooth muscle, skeletal and heart muscle, and
aminergic and peptidergic endocrine cells (25 37 ). In these typical
excitable cells, NaCh is responsible for the generation of electrical
signals, i.e. the generation of action potentials (38 ).
However, this may not be their sole function, since nonexcitable
cells, namely glial cells (34 ), have also been found to express NaCh.
The glial form expressed by Schwann cells is closely related to eNaCh
(
93% homology). The density of NaCh in glial cells and GC appear
also to be comparable (34 ). Moreover, GC and luteal cells in the ovary
are tightly coupled via gap junctions (see references in Ref. 39 ). Gap
junctions provide a low resistance shunt to adjacent cells, and
coupling will therefore prevent large membrane depolarizations. These
facts and our experimental results (e.g. most GC tested had
a membrane potential where the main portion of
Na+ channels would be in the inactivated state)
make it rather unlikely that GC or luteal cells are excitable.
Provided that generation of action potentials is not a role of eNaCh,
what is the function of eNaCh in GC and in the CL? To address this
question we took advantage of the fact that NaChs can be activated by
veratridine or blocked by TTX (25 37 38 40 ). Addition of TTX to
human GC, while causing no significant change in progesterone
production of these cells, induced an ultrastructurally highly
differentiated cellular phenotype with abundant mitochondria, which was
distinct from untreated control cells. Thus, functional TTX-sensitive
eNaChs appear to be present, although progesterone production alone did
not reflect this supposition. In contrast, pharmacological
activation of eNaCh by veratridine produced both a decrease in
progesterone release and conspicuous signs of cellular regression.
These included increases of secondary lysosomes, indicative of
autophagocytosis. Reduced progesterone could most likely be a direct
consequence of autophagy of cellular compartments involved in
steroidogenesis, such as mitochondria and smooth endoplasmic reticulum.
That these processes were indeed initiated and/or sustained by
activation of eNaCh on GC became clear from two types of experiments:
1) concomitant treatment of GC with TTX prevented the striking effect
of veratridine; 2) veratridine or TTX treatment of human epithelial
A431 carcinoma cells, which do not posses NaCh, induced no
ultrastructural change (no increase in lysosomes).
These results, in particular the morphological changes observed in
TTX-treated GC, allow the conclusion that eNaChs are present and
functional in GC. From our current point of view, this functionality
pertains to the possibility of persistent Na+
influx mediated by eNaCh. The membrane potential of most cells tested
was in a range where a possible window current would be maximal (-20
to -30 mV), with a probability of open channels of up to 4%.
Untreated cells tended to more negative V50
values of eNaCh activation with cultivation time, possibly resulting in
a slow increase of Na+ influx. Interestingly, hCG
treatment led to a shift of the resting membrane potential to more
positive values after 48 h and therefore would reduce persistent
Na+ current influx. We do not expect high levels
of Na+ influx to occur within the first days of
GC cultivation in the absence of hCG, because TTX, apart from its
morphological effects, provoked only a small and not statistically
significant increase in progesterone production (Fig. 8
). However, if
cells change their properties as described above, an increase in
Na+ influx can be expected at later time
points.
Clearly, we currently do not have information about the probability of
persistent Na+ fluxes to occur in luteal cells
in vivo, which express the eNaCh gene. Given this
possibility, however, how are these linked to regression of GC and
luteal cells? Support for such a link comes from our observations and
also from experiments performed in other species and organ systems. Our
ultrastructural studies performed on sections from the Rhesus monkey CL
show that similar events associated with eNaCh activation in
vitro (in GC) occur also in vivo. These include
lysosomal activity leading to autophagy of luteal cells. Both primary
and secondary lysosomes were present in monkey luteal cells on day 7 of
the luteal phase, i.e. in the functional CL. Primary and
secondary lysosomes were more readily detected around day 11 of the
luteal phase, and the number and size of secondary lysosomes were
increased in the regressing CL. In several species, including guinea
pig, pig, monkey, and human, lysosomes and/or increased lysosomal
activity or autophagy were previously described in the regressing CL
(20 21 22 23 ). In humans and nonhuman primates, luteolysis occurring at the
level of the luteal cell appears to differ from the processes reported
for the rat (see Introduction). The available reports
addressing this issue describe vacuolated cells and autophagy involving
lysosomes, e.g. in the marmoset CL (19 20 ). Thus, the
observed changes in GC and luteal cell morphology are corroborated by
other authors and mirror normal physiological processes.
That the family of NaCh, by altering intracellular ion concentrations,
are involved in the regulation of cell viability and forms of cell
death in a variety of cell types is clearly shown by several reports
(34 41 ). For instance, in glial cells it has been proposed (34 ) that
their NaCh may serve as a return pathway for Na+
ions, thus fueling the
Na+/K+ ATPase. Experimental
blockage of astrocyte NaCh by TTX produced a dose- dependent
reduction in glial cell viability (42 ). Such a possibility can almost
be ruled out for GC, in which TTX treatment resulted in the opposite
and preserved a structurally well differentiated cellular phenotype. In
neuronal cells, intracellular Na+ overload,
initiated by NaCh activity, has been linked to necrosis occurring in
cerebral ischemia or trauma (40 ). Unfortunately, a detailed picture of
the events leading to these changes in neurons can presently not be
given and certainly neuronal cell necrosis cannot be compared with the
regressive changes observed in GC and luteal cell, namely the
accumulation of secondary lysosomes and the lack of typical signs of
necrotic cell death or of apoptosis.
In summary, we propose the following hypothesis: The gonadotropins
LH/hCG negatively regulate both eNaCh gene expression and eNaCh
activity, as well as membrane potential of GC and presumably luteal
cells. LH/hCG thus act to prevent the observed deleterious effect after
expression and function of eNaCh on GC and luteal cells. This implies
that reduced LH values/or reduced accessibility/bioactivity of this
hormone to luteal cells causes the expression of active eNaChs in
luteal cells during the menstrual cycle. From the current point of
view, eNaChs allow persistent Na+ influx to
occur, which then initiates a process of luteal cell regression
involving autophagocytosis. The regulated expression and function of
these channels may represent an, as yet unknown, way by which
functional luteolysis occurs in the primate CL at the level of
individual cells. Ovarian NaCh may therefore be a key molecular element
in luteolysis.
Our findings could have implications for human physiology and human
diseases: NaChs are targets for various drugs, including local
anesthetics and systemically applied antiarrhythmics, as well as
antiepileptic drugs such as phenytoin (40 ). Our results raise the
question of whether alterations of CL function(s) may be a possible
consequence of systemic treatment with such substances in women
(43 ).
 |
MATERIALS AND METHODS
|
---|
Cell Culture Chemicals and Treatments
Follicular fluid containing granulosa cells was derived from
in vitro fertilization patients (44 ). The experimental
procedure and the use of the cells were approved by the local ethics
committee, and the women gave their written consent. Isolation and
culture of granulosa cells were performed as described previously (44 ).
Cells were seeded in DMEM/Hams F12 (1:1) medium with 10% FCS onto
Falcon culture dishes (Nunc, Wiesbaden, Germany), Labtek cell culture
chambers (Nunc), or on glass cover slides (for patch-clamp
experiments). All surfaces were coated with laminin
(Sigma, Deisenhofen, Germany), and cells were kept in
culture for up to 11 days (44 45 ). Culture medium was changed every 2
days. The human carcinoma cell line (A431) was obtained from Dr. M.
Haasemann (Munich, Germany) and used for morphological studies (46 ).
All chemicals used in stimulation experiments were purchased from
Sigma. For lysosomal dye studies, ultrastructural
examination, and progesterone release, cells were incubated for 24
h or 48 h with veratridine (5 and 50 µM),
TTX (5 and 50 µM), or human CG (hCG; 10
IU/µl). Ethanol was used to dilute veratridine and was therefore
added in a final concentration of 0.5% to cells not treated with
veratridine as an additional control.
Tissues
The care and housing of rhesus macaques (Macaca
mulatta) at the Oregon Regional Primate Research Center (ORPRC)
were previously described (47 ). Animal protocols and experiments were
approved by the Oregon Regional Primate Research Center Animal Care and
Use Committee, and studies were conducted in accordance with the NIH
Guide for the Care and Use of Laboratory Animals. Ovaries were
collected from rhesus macaques undergoing ovariectomy (see below) for
other purposes or were obtained at necropsy from the tissue
distribution program at the ORPRC (n = 3). In total, ovaries from
14 adult monkeys were examined. Upon collection, all tissues were
rapidly frozen on dry ice (for extraction of RNA) or immersed in
fixative for electron microscopy (see below). CL samples were obtained
as described previously (47 ). Adult female monkeys with regular
menstrual cycles were bled daily by saphenous venipuncture beginning on
day 8 after the onset of menses. Serum concentrations of estradiol
(E2) and progesterone were measured by RIA in the
Endocrine Services Laboratory at the ORPRC (48 ). The day of the
precipitous fall in circulating E2 levels after
the midcycle peak was designated day 1 of the luteal phase (48 ).
Corpora lutea were surgically removed from anesthetized monkeys on
different days of the luteal phase for RNA extraction (days 3, 10 and
14; total of 4 samples) or for fixation for electron microscopy (days
7, 11, 15, 16, 18; total of 7 samples), as previously described
(47 ).
Electrophysiology
Patch pipettes were manufactured from borosilicate glass
capillaries and fire polished on a DMZ-Universal puller (Engel,
Augsburg, Germany), with resistances between 3 and 7 M
. Seal
resistances typically ranged from 2 to 5 G
and were established
within 30 sec. Before rupture of the cell membranes, the potential was
clamped to -70 mV. All recordings were performed in the whole-cell
configuration at room temperature using an EPC-9 amplifier controlled
by PULSE (Heka, Lambrecht, Germany). Patch pipettes were filled with a
solution containing (in millimolar concentration): 140 KCl, 10 HEPES, 5
EGTA, 1 CaCl2, 1 MgCl2, (pH
7.4/KOH, free Ca2+ 100 nM). For
investigation of Na+ currents alone, a solution
was used containing (in millimolar concentration): 120 CsCl, 20 TEA-Cl,
5 EGTA, 0.5 CaCl2, 1 MgCl2,
10 HEPES (pH 7.4 with CsOH, free Ca2+ 10
nM). The bathing solution consisted of (in millimolar
concentration): 140 NaCl, 3 KCl, 1 CaCl2, 10
HEPES, 10 glucose (pH 7.4 with NaOH). For local and fast application of
TTX or hCG (Sigma, Deisenhofen, Germany) a seven-channel
superfusion system was used. Activation of Na+
currents was investigated with a cyclic pulse protocol (protocol 1).
Within each sequence the cell was hyperpolarized to -120 mV for 100
msec. Subsequently, a variable test potential from -90 to +60 mV was
applied. For investigation of steady-state inactivation, the cell was
clamped to the hyperpolarized potential, then to a variable
prepotential, and finally to the test potential of -20 mV (protocol
2). In both protocols, the time interval between sequences was 500
msec. For determination of TTX inhibition curves, constant test pulses
were applied in intervals of 1 sec. Cells tended to form branches and
were coupled by gap junctions. This was supported by the observation
that within a group of neighboring cells, the patched cell had an
apparently very low input resistance. To avoid this problem, isolated
cells were used for the experiments described.
RT-PCR
Preparation of total GC mRNA was done as described (44 ) using
the RNeasy kit from QIAGEN (Hilden, Germany), the acid
phenol-extraction method, as described previously (32 ), or by a cesium
chloride ultracentrifugation method (3 ). In addition, a commercial
human cDNA (2 µl), reverse transcribed from pooled adult ovarian
mRNA, was used for PCR (Invitrogen, DeSchelp, The
Netherlands). For reverse transcription, 200 ng of RNA together with
18-mer polydeoxythymidine primer and Moloneys murine leukemia virus
(Promega Corp., Mannheim, Germany) were incubated for
2 h at 37 C. For amplification of the sodium channel (NaCh)
subunit, primers were constructed to match common sequences of
different channel types from human (thyroid, brain) and rat (peripheral
nerve, brain; (5'-ATC GGA ATC TGA AGA CAG C-3', sense and 5'-CTG TGC
TCA TCA TCG GCA A-3', antisense). In some cases cyclophilin was
coamplified with the sodium channel using primers and conditions
described previously (49 ). PCR amplification was performed in a PTC-200
thermocycler (MJ Research, Inc., Watertown, MA) using
Taq polymerase (Promega Corp.) starting with a
94 C step for initial denaturation (5 min) followed by 35 cycles of 1
min annealing at 54 C, 2 min extension at 72 C, and 15 sec denaturation
at 94 C. PCR products were resolved on a 2% agarose gel and visualized
with ethidium bromide. For sequence analysis, they were either
sequenced directly using one of the primers or they were first
subcloned into the pGEMT vector (Promega Corp.).
Sequencing was performed as described previously (44 ) using a
fluorescence-based dideoxy sequencing reaction on an ABI model 373A DNA
sequencer (Perkin-Elmer Corp., Überlingen,
Germany).
Northern Blotting
Northern blotting was performed as described (50 ) using 10 µg
of total RNA. Riboprobes (eNaCh and actin) were prepared by in
vitro transcription using 32P-UTP and T7- or
SP6-RNA-polymerase (Promega Corp.). Transcripts were
purified with Nick-columns (Pharmacia Biotech, Freiburg,
Germany) and hybridized to the membrane containing the test RNA at 60 C
overnight. Subsequently, blots were washed five times at 65 C in
0.1 x SSC, 0.1% SDS and dried. Autoradiograms were developed
after 15 days. For densitometric measurements, blots were digitized
using an image documentation system (MWG-Biotech, Ebersberg, Germany).
Integrated optical densities were determined using a noncommercial
program. Densities obtained from the actin signal were used to
normalize the values obtained from eNaCh signals.
Fluorescence Microscopy: Lysosomal Dye
The density and size of lysosomes were analyzed in GC treated
with veratridine or TTX for 24 h using the LysoTracker L-7528
lysosomal dye (Molecular Probes, Inc., Eugene, OR). Cells
on glass cover slides were incubated with the dye (10 nM)
for 2 min at room temperature and subsequently observed and
photographed using an inverse fluorescence microscope (Axiovert
135TV;Carl Zeiss, Jena, Germany). The experiments were
repeated with three different preparations of cells.
Electron Microscopy
For ultrastructural studies, human A 431 carcinoma cells and GC
were incubated for 24 and 48 h with or without veratridine, TTX,
and hCG and were then fixed with 4% paraformaldehyde/0.5%
glutaraldehyde and postfixed with 4%
OsO4/potassium hexacyanoferrate (II). Another
control group, in which we tested whether veratridine effects can be
antagonized by TTX, consisted of GC pretreated with TTX (8 h) and
subsequent incubation with veratridine for 24 h. After embedding
in Epon, thin sections were cut, contrasted with uranylacetate
(2%)/lead citrate (2.7%) as described previously (51 52 ), and
examined with an EM10 electron microscope (Carl Zeiss).
Three different preparations of GC were examined. Small fragments of
nonhuman CL tissues obtained at different times of the luteal phase
(days 7, 11, 15, and 16, and day 18; total of 7) were also examined. In
this case, samples were immersed in 5% glutaraldehyde in 0.1
M cacodylate acid [pH 7.4, (52 )] and subsequently
processed as described above.
Progesterone Assay
Culture media from various stimulation experiments were
collected and frozen at -20 C until determination of progesterone
concentrations, using the Serozyme-M kit from BioChem (Freiburg,
Germany), as described (44 ).
The amount of protein was determined as described (44 ), and
progesterone values were expressed as nanograms/mg protein.
Statistics
For statistical analyses, unpaired and two-tailed t
tests were performed with the exception of progesterone values (Fig. 8
;
paired t test) and change of V50 with
time (F test against slope value of zero).
 |
ACKNOWLEDGMENTS
|
---|
We thank Mrs. G. Terfloth, Mrs. U. Fröhlich, Mrs. S.
Boddien, Mr. G. Prechtner, Mr. A. Mauermayer, and Mr. R. Grünert
for technical assistance and Dr. J. Grosse for the help with Northern
blotting.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Artur Mayerhofer, Anatomisches Institut, Technische Universität München, Biedersteiner Strasse 29, D-80802 München, Germany.
This work was supported by DFG grants Ma1080/121 and
Graduiertenkolleg 333, Volkswagen-Stiftung (A.M.), as well as by NIH
Grants HD-20869, HD-24870, and RR00163 (R.L.S., S.R.O.).
Received for publication October 5, 1999.
Revision received February 21, 2000.
Accepted for publication March 10, 2000.
 |
REFERENCES
|
---|
-
Zeleznik AJ, Benyo DF 1994 Control of follicular
development, corpus luteum function, the recognition of pregnancy in
higher primates. In: Knobil E, Neill JD (eds) The Physiology of
Reproduction. Raven Press, New York, pp 751782
-
Stouffer RL 1996 Corpus luteum formation and demise. In:
Adashi EY, Rock JA, Rosenwaks Z (eds) Reproductive Endocrinology,
Surgery, and Technology. Lippincott-Raven, New York, pp 263263
-
Duffy DM, Stewart DR, Stouffer RL 1999 Titrating luteinizing
hormone replacement to sustain the structure and function of the corpus
luteum after gonadotropin-releasing hormone antagonist treatment in
rhesus monkeys. J Clin Endocrinol Metab 84:342349[Abstract/Free Full Text]
-
Zeleznik AJ 1998 In vivo responses of the primate
corpus luteum to luteinizing hormone and chorionic gonadotropin. Proc
Natl Acad Sci USA 95:1100211007[Abstract/Free Full Text]
-
Guo K, Wolf V, Dharmarajan AM, Feng Z, Bielke W, Saurer S,
Friis R 1998 Apoptosis-associated gene expression in the corpus luteum
of the rat. Biol Reprod. 58:739746
-
Roughton SA, Lareu RR, Bittles AH, Dharmarajan AM 1999 Fas
and Fas ligand messenger ribonucleic acid and protein expression in the
rat corpus luteum during apoptosis-mediated luteolysis. Biol
Reprod 60:797804[Abstract/Free Full Text]
-
Gaytan F, Bellido C, Morales C, Sanchez-Criado JE 1998 Both
prolactin and progesterone in proestrus are necessary for the induction
of apoptosis in the regressing corpus luteum of the rat. Biol Reprod 59:12001206[Abstract/Free Full Text]
-
Billig H, Furuta I, Hsueh AJ 1994 Gonadotropin-releasing
hormone directly induces apoptotic cell death in the rat ovary:
biochemical and in situ detection of deoxyribonucleic acid
fragmentation in granulosa cells. Endocrinology 134:245252[Abstract]
-
Wyllie AH, Kerr JF, Currie AR 1980 Cell death: the
significance of apoptosis. Int Rev Cytol 68:251306[Medline]
-
Quirk SM, Cowan RG, Joshi SG, Henrikson KP 1995 Fas
antigen-mediated apoptosis in human granulosa/luteal cells. Biol Reprod 52:279287[Abstract]
-
Rodger FE, Fraser HM, Krajewski S, Illingworth PJ 1998 Production of the proto-oncogene BAX does not vary with changing in
luteal function in women. Mol Hum Reprod 4:2732[Abstract]
-
Fukaya T, Funayama Y, Muakami T, Sugawara J, Yajima A 1997 Does apoptosis contribute follicular atresia and luteal regression in
human ovary? Horm Res 48[Suppl 3]:2026
-
Funayama Y, Sasano H, Suzuki T, Tamura M, Fukaya T, Yajima A 1996 Cell turnover in normal cycling human ovary. J Clin
Endocrinol Metab 81:828834[Abstract]
-
Gaytan F, Morales C, Garcia-Pardo L, Reymundo C, Bellido C,
Sanchez-Criado JE 1998 Macrophages, cell proliferation, and cell death
in the human menstrual corpus luteum. Biol Reprod 59:417425[Abstract/Free Full Text]
-
Yuan W, Giudice LC 1997 Programmed cell death in human ovary
is a function of follicle and corpus luteum status [published erratum
appears in J Clin Endocrinol Metab 1998 Jan;83(1):240]. J
Clin Endocrinol Metab 82:31483155[Abstract/Free Full Text]
-
Shikone T, Yamoto M, Kokawa K, Yamashita K, Nishimori K,
Nakano R 1996 Apoptosis of human corpora lutea during cyclic luteal
regression and early pregnancy. J Clin Endocrinol Metab 81:23762380[Abstract]
-
Rodger FE, Fraser HM, Duncan WC, Illingworth PJ 1995 Immunolocalization of bcl-2 in the human corpus luteum. Hum Reprod 10:15661570[Abstract]
-
Fraser HM, Lunn SF, Cowen GM, Illingworth PJ 1995 Induced
luteal regression in the primate: evidence for apoptosis and changes in
c-myc protein. J Endocrinol 147:131137[Abstract]
-
Young FM, Illingworth PJ, Lunn SF, Harrison DJ, Fraser HM 1997 Cell death during luteal regression in the marmoset monkey
(Callithrix jacchus). J Reprod Fertil 111:109119[Abstract]
-
Fraser HM, Lunn SF, Harrison DJ, Kerr JB 1999 Luteal
regression in the primate: different forms of cell death during natural
and gonadotropin-releasing hormone antagonist or prostaglandin
analogue-induced luteolysis. Biol Reprod 61:14681479[Abstract/Free Full Text]
-
Paavola LG 1978 The corpus luteum of the guinea pig. III.
Cytochemical studies on the Golgi complex and GERL during normal
postpartum regression of luteal cells, emphasizing the origin of
lysosomes and autophagic vacuoles. J Cell Biol 79:5973[Abstract]
-
Gregoraszczuk EL, Sadowska J 1997 Lysosomal acid phosphatase
activity and progesterone secretion by porcine corpora lutea at various
periods of the luteal phase. Folia Histochem Cytobiol 35:3539[Medline]
-
Takenaka A 1981 An ultrastructural and cytochemical study of
human corpora lutea. Acta Obstet Gynaecol Jpn 33:907915[Medline]
-
Bulling A, Brucker C, Berg U, Gratzl M, Mayerhofer A 1999 Identification of voltage-activated Na+ and
K+ channels in human steroid-secreting ovarian
cells. Ann NY Acad Sci 868:7779[Free Full Text]
-
Klugbauer N, Lacinova L, Flockerzi V, Hofmann F 1995 Structure
and functional expression of a new member of the tetrodotoxin-sensitive
voltage-activated sodium channel family from human neuroendocrine
cells. EMBO J 14:10841090[Abstract]
-
Hille B 1992 Counting channels. In: Hille B (ed) Ionic
Channels of Excitable Membranes. ed 2. Sinauer Associates, Sunderland,
MA, p 329
-
French CR, Sah P, Buckett KJ, Gage PW 1990 A voltage-dependent
persistent sodium current in mammalian hippocampal neurons. J Gen
Physiol 95:11391157[Abstract]
-
Crill WE 1996 Persistent sodium current in mammalian central
neurons. Annu Rev Physiol 58:349362[CrossRef][Medline]
-
Toledo-Aral JJ, Moss BL, He ZJ, Koszowski AG, Whisenand T,
Levinson SR, Wolf JJ, Silos-Santiago I, Halegoua S, Mandel G 1997 Identification of PN1, a predominant voltage-dependent sodium channel
expressed principally in peripheral neurons. Proc Natl Acad Sci USA 94:15271532[Abstract/Free Full Text]
-
Sangameswaran L, Fish LM, Koch BD, Rabert DK, Delgado SG,
Ilnicka M, Jakeman LB, Novakovic S, Wong K, Sze P, Tzoumaka E, Stewart
GR, Herman RC, Chan H, Eglen RM, Hunter JC 1997 A novel
tetrodotoxin-sensitive, voltage-gated sodium channel expressed in rat
and human dorsal root ganglia. J Biol Chem 272:1480514809[Abstract/Free Full Text]
-
Dees WL, Hiney JK, Schultea TD, Mayerhofer A, Danilchik M,
Dissen GA, Ojeda SR 1995 The primate ovary contains a population of
catecholaminergic neuron-like cells expressing nerve growth factor
receptors. Endocrinology 136:57605768[Abstract]
-
Mayerhofer A, Smith GD, Danilchik M, Levine JE, Wolf DP,
Dissen GA, Ojeda SR 1998 Oocytes are a source of catecholamines in the
primate ovary: evidence for a cell-cell regulatory loop. Proc Natl
Acad Sci USA 95:1099010995[Abstract/Free Full Text]
-
Bukovsky A, Caudle MR, Keenan JA, Wimalasena J, Upadhyaya NB,
Van Meter SE 1995 Is corpus luteum regression an immune-mediated event?
Localization of immune system components and luteinizing hormone
receptor in human corpora lutea. Biol Reprod 53:13731384[Abstract]
-
Sontheimer H, Black JA, Waxman SG 1996 Voltage-gated
Na+ channels in glia: properties and possible
functions. Trends Neurosci 19:325331[CrossRef][Medline]
-
Mayerhofer A, Sterzik K, Link H, Wiemann M, Gratzl M 1993 Effect of oxytocin on free intracellular Ca2+
levels and progesterone release by human granulosa-lutein cells. J
Clin Endocrinol Metab 77:12091214[Abstract]
-
Schiffmann SN, Desdouits F, Menu R, Greengard P, Vincent JD,
Vanderhaeghen JJ, Girault JA 1998 Modulation of the voltage-gated
sodium current in rat striatal neurons by DARPP-32, an inhibitor of
protein phosphatase. Eur J Neurosci 10:13121320[CrossRef][Medline]
-
Catterall WA 1992 Cellular and molecular biology of
voltage-gated sodium channels. Physiol Rev 72:S15S48
-
Catterall WA 1995 Structure and function of voltage-gated ion
channels. Annu Rev Biochem 64:493531[CrossRef][Medline]
-
Mayerhofer A, Garfield RE 1995 Immunocytochemical analysis of
the expression of gap junction protein connexin 43 in the rat ovary.
Mol Reprod Dev 41:331338[CrossRef][Medline]
-
Taylor CP, Meldrum BS 1995 Na+ channels
as targets for neuroprotective drugs. Trends Pharmacol Sci 16:309316[CrossRef][Medline]
-
Bortner CD, Hughes FMJ, Cidlowski JA 1997 A primary role for
K+ and Na+ efflux in the
activation of apoptosis. J Biol Chem 272:3243632442[Abstract/Free Full Text]
-
Sontheimer H, Fernandez-Marques E, Ullrich N, Pappas CA,
Waxman SG 1994 Astrocyte Na+ channels are
required for maintenance of
Na+/K+-ATPase activity.
J Neurosci 14:24642475[Abstract]
-
Murialdo G, Galimberti CA, Magri F, Sampaolo P, Copello F,
Gianelli MV, Gazzerro E, Rollero A, Deagatone C, Manni R, Ferrari E,
Polleri A, Tartara A 1997 Menstrual cycle and ovary alterations in
women with epilepsy on antiepileptic therapy. J Endocrinol Invest 20:519526[Medline]
-
Mayerhofer A, Hemmings HCJ, Snyder GL, Greengard P, Boddien S,
Berg U, Brucker C 1999 Functional dopamine-1 receptors and DARPP-32 are
expressed in human ovary and granulosa luteal cells in
vitro. J Clin Endocrinol Metab 84:257264[Abstract/Free Full Text]
-
Mayerhofer A, Engling R, Stecher B, Ecker A, Sterzik K, Gratzl
M 1995 Relaxin triggers calcium transients in human granulosa-lutein
cells. Eur J Endocrinol 132:507513[Medline]
-
Haasemann M, Cartaud J, Muller-Esterl W, Dunia I 1998 Agonist-induced redistribution of bradykinin B2 receptor in caveolae.
J Cell Sci 111:917928[Abstract/Free Full Text]
-
Vandevoort CA, Molskness TA, Stouffer RL 1988 Adenylate
cyclase in the primate corpus luteum during chorionic gonadotropin
treatment simulating early pregnancy: homologous versus
heterologous desensitization. Endocrinology 122:734740[Abstract]
-
Stouffer RL, Dahl KD, Hess DL, Woodruff TK, Mather JP,
Molskness TA 1994 Systemic and intraluteal infusion of inhibin A or
activin A in rhesus monkeys during the luteal phase of the menstrual
cycle. Biol Reprod 50:888895[Abstract]
-
Mayerhofer A, Dissen GA, Costa ME, Ojeda SR 1997 A role for
neurotransmitters in early follicular development: induction of
functional follicle-stimulating hormone receptors in newly formed
follicles of the rat ovary. Endocrinology 138:33203329[Abstract/Free Full Text]
-
Mayerhofer A, Dissen GA, Parrott JA, Hill DF, Mayerhofer D,
Garfield RE, Costa ME, Skinner MK, Ojeda SR 1996 Involvement of nerve
growth factor in the ovulatory cascade: trkA receptor activation
inhibits gap junctional communication between thecal cells.
Endocrinology 137:56625670[Abstract]
-
Höhne-Zell B, Gratzl M 1996 Adrenal chromaffin cells
contain functionally different SNAP-25 monomers and
SNAP-25/syntaxin heterodimers. FEBS Lett 394:109116[CrossRef][Medline]
-
Mayerhofer A, Weis J, Bartke A, Yun JS, Wagner TE 1990 Effects
of transgenes for human and bovine growth hormones on age-related
changes in ovarian morphology in mice. Anat Rec 227:175186[Medline]