1 Department of Biochemistry, Intracellular
Ca2+ release channels such as
ryanodine receptors play crucial roles in the
Ca2+-mediated signaling that
triggers excitation-contraction coupling in muscles. Although the
existence and the role of these channels are well characterized in
skeletal and cardiac muscles, their existence in smooth muscles, and
more particularly in the myometrium, is very controversial. We have now
clearly demonstrated the expression of ryanodine receptor
Ca2+ release channels in rat
myometrial smooth muscle, and for the first time, intracellular
Ca2+ concentration experiments
with indo 1 on single myometrial cells have revealed the existence of a
functional ryanodine- and caffeine-sensitive Ca2+ release mechanism in 30% of
rat myometrial cells. RT-PCR and RNase protection assay on whole
myometrial smooth muscle demonstrate the existence of all three
ryr mRNAs in the myometrium:
ryr3 mRNA is the predominant subtype,
with much lower levels of expression for
ryr1 and
ryr2 mRNAs, suggesting that the
ryanodine Ca2+ release mechanism
in rat myometrium is largely encoded by
ryr3. Moreover, using intracellular
Ca2+ concentration measurements
and RNase protection assays, we have demonstrated that the expression,
the percentage of cells responding to ryanodine, and the function of
these channels are not modified during pregnancy.
calcium-induced calcium release; in situ hybridization; gene
regulation; smooth muscle; inositol trisphosphate
RYANODINE RECEPTORS (RyRs) located in the sarcoplasmic
reticulum (SR) membrane of muscle tissues have a predominant role in excitation-contraction (EC) coupling (40). In heart and some smooth
muscles, RyRs are the molecular basis for the
Ca2+-induced
Ca2+ release (CICR) mechanism that
contributes to mechanical activity (3, 40). However, in uterine smooth
muscle (myometrium), where spontaneous contractile activity is
dependent on spontaneous discharge of
Ca2+ action potentials (45), the
existence and role of CICR and, consequently, the involvement of the SR
in spontaneous contractions are still a matter of controversy.
In this connection, it is well known that during pregnancy the uterus
undergoes dramatic modifications of its form and function and displays
remarkable and essential changes in its electrical, mechanical, and
biochemical characteristics (2, 17, 21). For example,
1) the density of
Na+ channels and the number of gap
junctions increase during pregnancy (16, 29), and
2) mRNAs encoding voltage-dependent
Ca2+ channel (VDCC) subunits also
increase in the pregnant rat myometrium (44). Although little is known
about possible gestation-dependent expression and the role of RyRs,
recent studies suggest that CICR may play a role in the EC coupling in
rat myometrium at the end of gestation (39, 42).
Two major classes of Ca2+ channel
family are involved in the release of
Ca2+ from intracellular stores:
the inositol trisphosphate
(InsP3)-gated Ca2+ release channel
[InsP3 receptors
(InsP3Rs)] and RyRs (19,
41). Both comprise three main subtypes:
InsP3RI-III and RyR1-3,
respectively. RT-PCR experiments have revealed the expression of all
three InsP3R subtypes in the
myometrium during human pregnancy (31). Moreover, induction of
Ca2+ release occurs with direct
application of InsP3 to
permeabilized myometrial cells or purified myometrial SR (4, 31). In
addition, application of InsP3 to
permeabilized myometrial cell preparations induces a contractile
response (18, 38), suggesting a functional role for
InsP3R during EC coupling in the
myometrium. However, although
InsP3Rs play an important role in
Ca2+ homeostasis in the
myometrium, their expression does not change during pregnancy (31). In
contrast to InsP3Rs, little is
known regarding the expression and function of ryanodine-sensitive
Ca2+ release channels in the
myometrium. Although RT-PCR experiments have revealed the presence of
RyRs in human myometrium (24) and we have presented
electrophysiological evidence for a ryanodine-sensitive cation channel
in nonpregnant myometrium from rat (26), ryanodine fails to produce
force or intracellular Ca2+
concentration
([Ca2+]i)
transients in cultured myometrial cells from pregnant rats (1).
Moreover, caffeine, generally accepted to be a universal activator of
CICR, is unable to release Ca2+
from internal stores or to evoke contractions or
[Ca2+]i
transients in human and rat myometrium (1, 15, 35, 37). Taken together,
these data are difficult to reconcile. One of the reasons for the
disparities in the cited studies may be differences in experimental
conditions, e.g., human vs. animal tissue, cultured vs. fresh cells,
and nonpregnant vs. pregnant myometrium.
We have used a combination of molecular biology [RT-PCR and RNase
protection assays (RPAs)] and measurement of
[Ca2+]i
(fluorophore dye indo 1 system) to examine freshly isolated rat
myometrial cells to 1) identify
which ryr genes are expressed in
myometrial smooth muscle, 2)
determine whether their mRNA transcripts are differentially expressed
over the course of pregnancy, and 3)
characterize RyR function during pregnancy.
Collection of tissues.
Experiments were performed on uteri from Sprague-Dawley and Wistar
female rats. Animals were anesthetized with ether and killed by
cervical dislocation.
Preparation of isolated myometrial cells.
Uteri were removed and placed in a physiological salt solution (PSS)
composed of (in mM) 130 NaCl, 5.6 KCl, 2 CaCl2, 1 MgCl2, 11 glucose, and 10 HEPES,
with pH adjusted to 7.4 with NaOH. The uterine horn was opened
longitudinally, and under binocular control the endometrium and the
circular muscle layer were removed. The remaining longitudinal muscle
layer was cut into several pieces (1 × 1 mm), incubated for 10 min in Ca2+- and
Mg2+-free PSS, and then incubated
in Ca2+- and
Mg2+-free PSS containing 0.09%
(wt/vol) collagenase (type CLS1, Worthington), 0.045% pronase (type E,
Sigma Chemical), and 3% (wt/vol) BSA (Sigma Chemical) at 37°C for
two successive periods of 25 min. Then the solution was removed and the
myometrial pieces were incubated again in a fresh enzyme-free solution
and triturated with a fire-polished pasteur pipette to release cells.
Cells were stored on glass coverslips at 4°C in PSS containing 0.8 mM Ca2+ and used on the same day.
Measurement of
[Ca2+]i.
We used the Ca2+-sensitive
fluorophore indo 1 to assess dynamic changes in
[Ca2+]i
in individual myometrial cells. Cells were loaded with 1 µM indo 1-AM
(Calbiochem) for 30 min at room temperature (20 ± 0.5°C). Coverslips containing indo 1-loaded cells were then washed for 25 min
with fresh PSS to remove extracellular indo 1-AM.
[Ca2+]i
was estimated from the indo 1 fluorescence using single-wavelength excitation (360 ± 10 nm) and dual emission (405 ± 10 and 480 ± 10 nm) (13). The recording apparatus included a Nikon (Diaphot 300) inverted microscope fitted with epifluorescence (×40 oil immersion objective). The intensities of transmitted light from a
window slightly larger than the cell were simultaneously recorded by
two photometers (model P100, Nikon), and single-photon currents were
converted to voltage signals. Signals at each wavelength were digitized
and stored on a personal computer by using a PC-Lab Card 812PG
interface, with sampling at 17 Hz. The ratio (R = F405/F480, where F405 and
F480 represent fluorescence at 405 and 480 nm) was calculated on-line and displayed with the two voltage
signals on a monitor.
[Ca2+]i
was estimated from the ratio of the fluorescence (12), with use of a
specific calibration for indo 1 determined within myometrial cells, as
previously described (13). Agonists (ACh, caffeine, ryanodine, and
oxytocin; Sigma Chemical) were applied to the recorded cell by pressure
ejection from a glass pipette for the period indicated on the records.
No change in
[Ca2+]i
was observed during control ejection of PSS. Each record of [Ca2+]i
response to agonists was obtained from a different cell, and each type
of experiment was repeated for the number of cells
(n) indicated. No differences were
seen in the resting
[Ca2+]i
value or in the agonist-induced
[Ca2+]i
response between the two rat strains.
Tissue collection and RNA isolation.
The following groups were compared: nonpregnant (random cycling) and
timed-pregnant rats at 10, 16-17, 18-19, 20-21, and 22 days postcoitus (nonlabor), 22 days during parturition, and 1 or 3 days
postpartum. Uterine horns were removed and opened along the line of
placental attachment. Pups and placenta were removed, and the
endometrium was carefully scraped. Animals were killed during
parturition after delivery had initiated, and usually when at least two
pups had been delivered. The myometrium was then washed to remove all
traces of blood, cut in small fragments, directly frozen in liquid
nitrogen, and stored at Oligonucleotide primers, RT, and PCR amplification.
Nondegenerate sense and antisense primers (Oswel DNA Service,
Southampton, UK) were designed as follows:
1)
ryr1a
5'-GGAAAGAGATTGTGAACCTG-3' and
ryr1b
5'-CTGTCAGGAATGGAACCACT-3' correspond to nucleotides 1673-1692 and 2139-2158, respectively, of the pig
ryr1 cDNA (10, 22).
2)
ryr2a
5'-TCTGAATTCATGACCCTCCTGCACTTC-3' and
ryr2b
5'-CACTCTAGATGCTGACTCTGGAACTTC-3', based on the amino acid
sequence between amino acids 4293 and 4479 of the rabbit type 2 sequence (34), amplify a PCR product of 582 bp. A further nested primer
set, based on the mouse sequence, was used to amplify an
ryr2 PCR fragment of 454 bp:
ryr2c
5'-ATGTGTATGAAGCTGCCTCAACTT-3' and
ryr2d
5'-GGGCAGCCTGGTGGAAGGTGC-3'.
3)
ryr3a
5'-GTGTCAAAGTAGTCATTGCCAA-3' and
ryr3b
5'-ATCCTGTCATCTGTAACTCACAA-3' were designed to amplify the
cDNA corresponding to amino acids 4648-4708 of the mink type 3 protein sequence (28), giving a PCR product of 445 bp. In some
experiments a further pair of primers identical to that described by
Miyatake et al. (30) was used. 4)
InsP3RIa
5'-GTGATCAAGAAAGCCTACATG-3' and
InsP3RIb
5'-TAAACGAAATGCTGCTCCAGA-3' were designed to amplify a cDNA encoding amino acid residues 2117-2275 of the human
InsP3RI protein sequence (32),
giving a PCR product of 467 bp.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70°C for later RNA preparation.
Total RNA from skeletal muscle, uterus, and whole brain was isolated as
described previously (7), resuspended in RNase-free
H2O, and stored at
70°C. All the samples had intact 18S and 28S RNAs, as judged
by ethidium bromide staining after agarose gel electrophoresis. Rat
brain, cerebellum, and skeletal muscle were handled in the same manner
to provide positive control RNA for PCR and RPA experiments. No
differences were seen in ryr mRNA
expression between the two rat strains.
Cloning and sequencing of RT-PCR products. PCR products were subcloned into pGEM-(T) or pGEM-(T) easy (Promega) by overnight ligation at 15°C in 30 mM Tris · HCl (pH 7.8), 10 mM MgCl2, 10 mM dithiothreitol, 0.5 mM ATP, and 3 units of T4 DNA ligase. Subclones of each ryr PCR product were sequenced on both strands with use of a Sequenase II sequencing kit (Amersham).
RPA.
RPAs were performed on three to six uterine samples for each date of
pregnancy by use of the HybSpeed RPA kit (Ambion) according to the
manufacturer's instructions. Negative controls contained yeast RNA
instead of rat RNA. Rat whole brain RNA was used as a positive control
and was processed at the same time as the different uterine samples.
Rat
InsP3RI
and ryr1, ryr2, and
ryr3 partial cDNA clones were
linearized and transcribed in vitro using
[-32P]UTP (800 Ci/mmol; Amersham). A rat actin cRNA (pTRI-
-Actin, Ambion) was also
synthesized and used as an internal control for assay variability.
After purification by passage over Nick columns (Pharmacia), cRNA
probes (9 × 105 cpm) were
hybridized to total myometrial (50 µg) or whole brain RNA (25 µg)
in hybridization buffer (Ambion) for 10 min at 68°C. Free cRNA
(nonhybridized) was removed by digestion with a 1:25 dilution of a
mixture of RNase A (1 mg/ml) and RNase T1 (20,000 U/ml) for 30 min at
37°C. Samples were ethanol precipitated and resuspended in loading
buffer for separation on a 4% or 5% (wt/vol) polyacrylamide gel
containing 8 M urea. The gels were dried and exposed to X-ray film
(Dupont) at
70°C for up to 2 wk with use of intensifying
screens. Quantitation was carried out by densitometry with use of a
computer-driven image analysis system (Seescan). The ratio of
InsP3RI
to
-actin or
ryr to
-actin in brain was arbitrarily set
to 1, and results from myometrial RNA were expressed relative to brain.
Statistics. Data were assessed by ANOVA followed by a post hoc test. Significance was set at P < 0.05. Values are means ± SE.
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RESULTS |
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RyR Ca2+
release channels are present and functional in single myometrial cells
at 16-17 days of pregnancy.
Using drugs known to act on these channels, including ryanodine and
caffeine, we first assessed the function of RyRs in myometrial cells.
Microejection of 10 or 50 µM ryanodine near the cell for 30 s evoked
a transient increase in
[Ca2+]i,
which rose to 135 ± 8 nM (n = 11)
and 213 ± 20 nM (n = 43), respectively,
from a resting value of 73 ± 4 nM (n = 220; Fig. 1A).
|
|
|
Effect of pregnancy on ryanodine- and caffeine-induced
[Ca2+]i
response.
We investiged whether RyR function changes during pregnancy by
determining the effect of ryanodine and caffeine on
[Ca2+]i
in myometrial cells isolated at 16-17 and 20-21 days of
pregnancy. In cells from the late-stage pregnancy (20-21 days),
the resting value of
[Ca2+]i
was slightly increased: 102 ± 4.7 nM
(n = 160) for 20-21 days compared
with 72 ± 1 nM (n = 220) for
16-17 days (P < 0.05). However, neither the percentage of responding cells nor the amplitude of the
[Ca2+]i
responses induced by ryanodine (10-50 µM) or caffeine
(0.5-5 mM) changed between the two stages of pregnancy (Fig.
4).
|
Isolation of partial cDNAs encoding rat RyRs. Partial cDNAs encoding rat RyRs were obtained from tissues known to express the different isoforms. Thus RT-PCR was carried out on total RNA from rat skeletal muscle RNA (ryr1 and ryr3) and whole brain RNA (ryr2). For ryr2, a nested PCR was performed. This procedure amplified 486-, 454-, and 445-bp fragments encoding ryr1, ryr2, and ryr3 cDNA, respectively (results not shown). The PCR products were subcloned, and their identity was verified by DNA sequencing. The sequence of the rat skeletal muscle ryr1 cDNA was 90 and 91% identical to the previously reported pig and rabbit nucleotide sequences, respectively (22). The ryr2 cDNA from whole rat brain was 85 and 93% identical to human and mouse ryr2 nucleotide sequences, respectively (27). The nucleotide sequence of the partial ryr3 cDNA was 91% identical to the human ryr3 nucleotide sequence and 100% identical to the mink RyR3 amino acid sequence (28).
Analysis of RyR channel expression by RT-PCR.
The ryr1-, ryr2-, ryr3-, and
InsP3RI-specific
PCR primers were used to screen cDNA reverse transcribed from total
myometrial RNA isolated from nonpregnant, pregnant (10, 16, 19, and 21 days or during parturition), and postpartum rats (1 day postpartum). Although RT-PCR is not quantitative, the technique gave a clear indication of the presence of the corresponding
ryr mRNA in the sample tested (Fig.
5). Total RNA from whole brain [which
expresses all the receptors (11, 27)] was used as a positive
control for the PCR, and samples where cDNA was replaced by
H2O were used to test for any
contamination (results not shown). All three known rat
ryr mRNAs, as well as
InsP3RI
mRNA, were represented in each of the myometrial RNA samples, as well
as in the sample from rat whole brain (Fig. 5). All PCR amplification
products were as easily detectable on ethidium bromide-stained agarose
gels as rat brain RT-PCR products, suggesting that these genes are well
expressed in rat myometrium. Moreover, with 30 cycles of PCR, no
important differences were detected between the different myometrial
samples, suggesting that ryr or
InsP3RI
mRNA expression does not change dramatically during pregnancy.
Furthermore, little sample-to-sample variability was detected (Fig. 5).
RT-PCR with specific primers designed to differentially amplify the
short and the long form of ryr3 (30)
shows that the long form (expected to be caffeine sensitive) is
expressed in the myometrium from pregnant rat (16, 19, 20, and 21 days
and during delivery) and nonpregnant rat (results not shown).
|
Quantitative analysis of RyR channel expression by RPA.
RNA probes complementary to ryr1, ryr2,
ryr3, and
InsP3RI
mRNA were each used in an RPA together with a rat
-actin cRNA to control for assay
variability (Fig. 6). For quantitation of
each ryr subtype and
InsP3RI,
the level of mRNA was expressed as a ratio to
-actin mRNA and relative to the
ratio of ryr to
-actin or
InsP3RI
to
-actin mRNA seen in rat brain.
Although the expression of
- and
-actin changes during
pregnancy,
-actin mRNA remains constant (6). The level of
InsP3RI
mRNA expression in rat myometrium was high (between 50 and 60% of
InsP3RI/
-actin
in rat brain; Fig. 6A). Of the three
subtypes, ryr3 has the highest level
of mRNA expression in the myometrium (50-75% of
ryr3/
-actin in rat brain; Fig.
6C).
ryr2 mRNA was barely detectable in the
myometrium, even after 2 wk of exposure, and was present at only 25%
of the ryr2/
-actin content of brain
(Fig. 6B). In myometrial samples, ryr1 mRNA was below the level of
detection of this technique. Expression of
InsP3RI,
ryr2, and ryr3 mRNA
remains constant from 10 days of pregnancy to 3 days after delivery and
does not change from the nonpregnant level (Fig. 6).
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DISCUSSION |
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The present work demonstrates that ryanodine-sensitive Ca2+ release stores do exist in rat myometrial smooth muscle cells. Moreover, examining functional RyR protein with use of [Ca2+]i measurements in single myometrial cells and expression of their corresponding mRNAs in whole myometrial smooth muscle, we observed that the expression and the function of these channels did not change during pregnancy.
Ryanodine- and caffeine-induced Ca2+ release in single myometrial cells. Our [Ca2+]i experiments have demonstrated the existence of a ryanodine- and caffeine-sensitive Ca2+ release mechanism in the rat myometrium. This finding confirms previous reconstitution of a ryanodine-sensitive Ca2+ release channel in that preparation (26). However, the present study is at variance with studies in which no effect of ryanodine or/and caffeine was seen on [Ca2+]i in cultured rat myometrial cells (1). A possible reason for this is that, in the study of Arnaudeau et al. (1), the myometrial cells had been cultured at least overnight and in some cases for several days before exposure to ryanodine and caffeine, and the expression of ryr mRNA may have been altered over the period in culture. It is well known that cell culture results in changes in phenotype, and it has been demonstrated in vascular smooth muscle that ryr mRNA and caffeine-sensitive [Ca2+]i responses are lost during the first (proliferative) stage of culture of smooth muscle (23, 36). Also, using an insufficient number of tested cells may give misleading results. Interestingly, we have observed ryanodine- and caffeine-induced [Ca2+]i responses in only ~30% of myometrial smooth muscle cells, as discussed below. The finding that only a subset of myometrial cells is sensitive to ryanodine and caffeine is of particular interest for the understanding of uterine pharmacology. This characteristic could explain why we and others have not observed contractile responses to caffeine in rat myometrium (18, 35). Caffeine has been shown to exert a potent inhibitory effect (relaxant action) on mechanical activity in a variety of smooth muscles, including myometrium (37). This relaxant action is due to a decrease in [Ca2+]i, as observed in 35% of cells in the present study, and to a decrease in the Ca2+ sensitivity of the contractile apparatus (35, 37). The decrease in [Ca2+]i is mediated, at least in part, by the methylxanthine-related inhibitory effect of caffeine on cyclic nucleotide-dependent phosphodiesterases (9), resulting in the stimulation of Ca2+ extrusion and Ca2+ storage after activation of plasmalemmal and SR Ca2+ pumps (8, 20). It is thus probable that, in the whole uterine muscle, the Ca2+ release effect of caffeine operating in a subset of cells is masked by the overall relaxant effect.
ryr mRNA subtypes in myometrium. The large size of mRNAs encoding ryr isoforms (>16 kb) and the low level of expression make quantitation difficult (e.g., by Northern blotting). However, using RT-PCR and RPAs, we have shown the presence of the three known ryr isoforms in rat myometrium during pregnancy and we have demonstrated that ryr3 mRNA is the major ryr mRNA expressed in myometrial tissues, whereas ryr1 is barely detectable. Using a cDNA probe corresponding to part of ryr3 cDNA, Hakamata et al. (14) reported expression of ryr3 mRNA in rabbit myometrium by Northern blotting. Furthermore, caffeine-insensitive Ca2+ release has been reported in rat myometrium (35), consistent with the presence of RyR3 in this tissue (mink RyR3 is also insensitive to caffeine). However, in recent experiments in which a full-length cDNA encoding the rabbit uterine RyR3 expressed in HEK-293 cells was used, the encoded Ca2+ release channel protein was sensitive to ryanodine and caffeine (5), suggesting that alternatively spliced mRNA variants (possibly with a premature termination codon) may be responsible for the different caffeine sensitivity of RyR3 in this tissue. However, our primers spanned the alternatively spliced exon; inasmuch as we only detected ryr3 mRNA, which included the alternatively spliced exon in the myometrium, this rules out the possibility that the alternatively spliced variant described by Miyatake et al. (30) is responsible for differential sensitivity to caffeine in the myometrium. Therefore, we can speculate that in rat myometrium the main Ca2+ release mechanism is dependent on RyR3, which can be activated by ryanodine and caffeine. However, although we did not detect any alternatively spliced mRNA variants with our RT-PCR, we cannot exclude their presence in other parts of this gene. ryr1 and ryr2 mRNAs are also expressed in the myometrium, but at a very low level, and we do not know whether these ryr mRNAs (if indeed they are translated) are coexpressed in the same cells as RyR3. As each Ca2+ release channel has a preferred mechanism of activation (e.g., Ca2+, voltage, or cyclic ADP-ribose) and can be activated by different stimuli, expression of more than one type of RyR in the myometrium could facilitate very precise regulation of [Ca2+]i in myometrial cells.
Effects of gestation on the myometrium.
Pregnancy is associated with changes in uterine structure and function
to accommodate the developing fetus and to prepare for parturition. The
molecular basis for these changes is poorly understood but must
certainly be linked to changes in gene expression. RT-PCR and
differential display techniques have been used to identify mRNAs for
which the level of expression changes during pregnancy (6). For
example, the expression of gap junctions,
Na+ channels, VDCC, and
cytoskeletal and matrix proteins is sensible to the gestational state
and seems to increase toward parturition (6, 16, 29, 44). In our study
the three ryr mRNA subtypes are
expressed in all myometrial samples (nonpregnant, pregnant, delivery,
and postpartum states), and microspectrofluorimetry has not revealed a
gestational modulation of the ryanodine or caffeine
[Ca2+]i
response in rat myometrium. This absence of gestational control of the
expression of Ca2+ channel release
has also been seen for the three subtypes of the
InsP3R (31) and for the three RyRs
in human myometrium (C. Martin, K. E. Chapman, S. Thornton, and R. H. Ashley, unpublished observations). Clearly, however, inasmuch as the
whole uterus undergoes an enormous gain in size during pregnancy, the
total expression of ryr mRNA in the
uterus will increase to reflect the increased number of cells. However,
the ratio of ryr to
-actin did not change. The only
change that we have detected in our preparation is a small increase of
the
[Ca2+]i
from 72 to 101 nM between 16-17 days and 20-21 days of
pregnancy. Using estrogen-treated rats to mimic a real pregnancy, Osa
(33) also observed an elevated basal
[Ca2+]i
in isolated myometrial cells from estrogen-treated compared with
nonpregnant rats. This
[Ca2+]i
change can be triggered by membrane depolarization occurring near term,
which brings the resting potential of the myometrial cell close to the
threshold of VDCC activation, or by the opening of receptor-operated
channels under hormonal stimuli, allowing Ca2+ entry (33).
Importance of RyR3 in the myometrium during parturition. We have demonstrated that the myometrium contains caffeine-sensitive Ca2+ stores. However, as only 30% of the cells in rat myometrium are caffeine and ryanodine sensitive, the physiological function of these stores is unclear. In this regard, it is worth noting that transgenic mice homologous for a targeted disruption of the ryr3 gene show normal growth and reproduction (43). However, it is possible that caffeine-sensitive stores are involved in the amplification of other Ca2+ mechanisms, such as InsP3-induced Ca2+ release or receptor-mediated Ca2+ influx. Contractions of the myometrium at the end of the pregnancy are important for the normal functions of the organ, and the cells containing RyR may play a pacemaker role by initiating the contractions needed for the delivery.
In conclusion, our demonstration of the existence of a functional ryanodine- and caffeine-sensitive Ca2+ release mechanism in 30% of rat myometrial cells is intriguing. It demonstrates that rat myometrial cells are not a homogeneous population and suggests that there are functional differences between the cells in terms of their contractile function. It raises important questions as to the main role of RyRs in the myometrium and, in the long term, may contribute to improvements in the clinical management of uterine dysfunction during pregnancy and childbirth. ![]() |
ACKNOWLEDGEMENTS |
---|
This research was supported by the Wellcome Trust, Institut National de la Santé et de la Recherche Médicale Grant CRI 9806, and Conseil Régional d'Aquitaine Grant 96031117. J.-M. Hyvelin is a recipient of an Agence de l'Environnement et de la Maitrise de l'Energie studentship.
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FOOTNOTES |
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The DNA sequences reported in this paper have been deposited in the GenBank/European Molecular Biology Laboratories database (accession nos. AF112256 and AF112257).
Present address of R. H. Ashley: Membrane Biology Group, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. Martin, Molecular Endocrinology, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK (E-mail: Cecile.Martin{at}ed.ac.uk).
Received 11 March 1999; accepted in final form 28 April 1999.
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