Intracellular mechanisms underlying prostaglandin
F2
-stimulated phasic myometrial
contractions
Mark
Phillippe,
Trevania
Saunders, and
Andrew
Basa
Department of Obstetrics and Gynecology, University of Chicago,
Chicago, Illinois 60637
 |
ABSTRACT |
These studies sought to test the hypothesis that
prostaglandin F2
(PGF2
)-stimulated phasic
myometrial contractions are characterized by the activation of the
phosphatidylinositol-signaling pathway resulting in the generation of
cytosolic calcium oscillations. For the experiments described in this
report rat myometrial tissue was used, after the tissue was loaded with
fura 2, to perform cytosolic calcium imaging studies and to perform
computer-digitalized in vitro isometric contraction studies. Consistent
with the above hypothesis, the cytosolic calcium-imaging studies
demonstrated PGF2
-stimulated
cytosolic calcium oscillations occurring simultaneously with phasic
contractions. The in vitro isometric contraction studies confirmed that
previously reported inhibitors of the phosphatidylinositol-signaling
pathway and cytosolic calcium oscillation mechanisms resulted in
significant inhibition of
PGF2
-stimulated phasic
myometrial contractions. In summary, these studies have provided
substantial support for the hypothesis that
PGF2
-stimulated phasic
myometrial contractions are generated by intracellular signaling
mechanisms involving activation of the phosphatidylinositol-signaling pathway and the production of cytosolic calcium oscillation-like phenomena.
cytosolic calcium oscillations; inositol phosphates; intracellular
calcium; phosphatidylinositol signaling pathway
 |
INTRODUCTION |
CYTOSOLIC CALCIUM oscillations have been previously
reported to occur in many types of excitable and nonexcitable cells in response to stimulation by various hormones and neurotransmitters; these reports have also confirmed that the phosphatidylinositol (PI)-signaling pathway plays a significant role during the generation of these calcium oscillations (2, 30). During these phenomena, receptor-activated stimulation of phosphoinositide-specific
phospholipase C (PI-PLC) has been shown to result in increased
production of inositol phosphates, especially inositol
1,4,5-trisphosphate (IP3), which
leads to the release of sequestered intracellular calcium. Subsequently, cycles of calcium release and reuptake by the
intracellular stores appear to result in sustained oscillations of
cytosolic calcium. Additional biochemical events that have been
reported to contribute to these phenomena include activation of
calcium-induced calcium release (CICR) from
IP3-insensitive calcium stores,
influx of extracellular calcium through membrane calcium channels,
negative feedback by protein kinase C, and possible oscillations in
IP3 production (2, 30).
Several recent reports have demonstrated that phasic smooth muscle
contractions, especially those occurring in female genital tract smooth
muscle, occur simultaneously with repetitive transients (or
oscillations) of cytosolic calcium (7, 8, 19, 23, 24, 28). These smooth
muscle calcium oscillations appear to be similar, if not identical, to
the cytosolic calcium oscillations observed in excitable and
nonexcitable cells as described above. Previous reports from our
laboratory have confirmed that activation of the PI-signaling pathway
using various agonists, including oxytocin, aluminum fluoride (a G
protein activator), potassium chloride (at concentrations of 10-30
mM), ionomycin, and BAY K 8644, results in the generation of phasic
myometrial contractions (21-24). Consistent with classic cytosolic
calcium oscillation phenomena, agonist-stimulated phasic myometrial
contractions were found to be markedly suppressed by
1) enzyme inhibitors of PI-PLC, 2) membrane permeant inhibitors of
intracellular calcium uptake and release,
3) phorbol ester activation of
protein kinase C, and 4) inhibition
of extracellular calcium influx (21-24).
Until recently, the signal transduction mechanisms underlying
prostaglandin stimulation have been both unclear and controversial. Several reports have confirmed that prostanoids bind to cell surface membrane receptors; to date, one prostaglandin F (PGF) membrane receptor and four prostaglandin E (PGE) membrane receptors have been
cloned and sequenced (18, 27). The PGE receptors appear to use multiple
intracellular signaling pathways (18), whereas the PGF receptor appears
to predominantly activate the PI-signaling pathway (27). Although
previous reports have confirmed inositol phosphate production in
response to PGF2
stimulation of
uterine myometrial cells (5, 13, 15, 20), the role of this signaling pathway during PGF2
-stimulated
phasic myometrial contractions has not been clearly defined. The
studies described in this report were undertaken to test the hypothesis
that PGF2
-stimulated phasic
myometrial contractions are generated by intracellular signaling
mechanisms involving activation of the PI-signaling pathway and
the production of cytosolic calcium oscillation-like phenomena.
 |
MATERIALS AND METHODS |
For these experiments, myometrial tissue was obtained from mature,
proestrus/estrus Sprague-Dawley white rats, using a protocol approved
by the Institutional Animal Care and Utilization Committee of the
University of Chicago. Under pentobarbital sodium (100 mg/kg)
anesthesia, the uteri were surgically removed, rinsed in normal saline,
then placed in Earle's buffered salt solution [EBSS; (in mM) 117 NaCl, 1.8 CaCl2, 5.3 KCl, 0.8 MgSO4, 1 NaH2PO4,
26.2 NaHCO3, and 5.6 glucose, pH
7.4] continuously bubbled with 95% O2-5%
CO2 .
The cytosolic calcium imaging studies were performed using the
longitudinal fiber layer of the myometrium, as previously reported (23,
24). Specifically, partial thickness myometrial strips (~4 × 6 mm) were prepared with the use of a dissecting microscope while the
tissue was kept in freshly aerated EBSS. Subsequently, the strips were
rinsed in a
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)buffered physiological salt solution [(in
mM) 150 NaCl, 2 CaCl2, 4 KCl, 1 MgCl2, 5.6 glucose, and 5 HEPES,
pH 7.4] aerated with O2 and
then loaded with fura 2, a ratiometric fluorescent intracellular
calcium-indicator dye. The loading incubations in freshly oxygenated
HEPES-buffered salt solution containing 2.5 µM fura 2-AM (the
membrane-permeant ester form of fura 2) along with 0.008% pluronic
acid were performed for 18-22 h at room temperature. Subsequently,
the myometrial tissue was pinned to parallel rubber strips attached to
the bottom of a heated perfusion dish (Delta-T Culture Dish System,
Biotechs, Butler, PA) mounted on a Nikon TMS inverted microscope
modified for epifluorescence
imaging.
To simultaneously determine force generation, the myometrial strips
were attached to a horizontal isometric force transducer (Kent
Scientific, Litchfield, CT) using stainless steel hooks. Before the
initiation of the experiments, the tissue was perfused in the dish with
fresh buffer at room temperature for at least 20-30 min to
facilitate completion of the hydrolysis of the ester form of the fura
2. During the subsequent calcium imaging experiments, the tissue was
continuously perfused with oxygenated buffer at 30°C. The low
temperature was used to decrease the rate of extrusion of the
fluorescent indicator dye; at this temperature spontaneous activity of
the muscle was uncommon. Tissue perfusion studies were performed using
10 nM or 0.5 µM PGF2
with and
without various inhibitors, including
2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC) and neomycin [reported inhibitors of PI-PLC (9, 11, 14,
21)], thimerosal [a biphasic modulator of
IP3-sensitive calcium release and
uptake (4, 26)], adenine and ruthenium red [reported
inhibitors of CICR (6, 22, 25)], and nifedipine [an L-type
calcium channel blocker (29)]. The fura 2 cytosolic calcium
measurements were performed using a video-based digital calcium imaging
system (Intracellular Calcium Imaging, Cincinnati, OH) setup for dual
excitation at 340 and 380 nm and emission at 505 nm. The calcium
results were reported as the relative fluorescent intensities of the
340 and 380 images and as the 340-to-380 ratio (340/380). The calcium
transients stimulated in response to oxytocin were compared with those
produced in response to a brief pulse of KCl (50 mM).
The in vitro isometric contraction studies were performed at 37°C
using longitudinal strips of uterine tissue (6-8 mm relaxed length) in 50-ml muscle baths, as previously reported from our laboratory (21, 22). Individual strips from individual animals were
used for each of the studies described in this report. Cumulative dose-response studies were performed using
PGF2
(1 nM to 1 µM) with and
without NCDC (80-120 µM), neomycin (1-5 mM), thimerosal (100-500 µM), adenine (1-5 mM), and nifedipine (1 µM).
The effects of these inhibitors were compared with control studies
performed using comparable volumes of vehicle alone. For the vehicle
control studies performed using ethanol or dimethyl sulfoxide (DMSO), the maximal concentration of ethanol in the muscle bath was 0.2 µl/ml, and the maximal concentration of DMSO was 2.4 µl/ml. To further evaluate the effects of extracellular calcium on
PGF2
-stimulated phasic
contractions, additional studies were performed during which the EBSS
buffer was rapidly replaced with preoxygenated, prewarmed calcium-free
EBSS buffer containing 2 mM ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA).
The analog data generated by the isometric contraction transducers were
digitalized and then analyzed to determine total contractile activity
(based on the area under the contraction curve) for 5-min intervals, as
previously reported from our laboratory (21, 22). Subsequently, the
contraction data were normalized for tissue cross-section area and
reported as the percentage of spontaneous or
PGF2
-stimulated contractile
activity. All studies were performed using 4-12 replicates, as
noted. Statistical analysis was performed using Kruskal-Wallis one-way
analysis of variance on ranks, the multiple comparisons
Student-Newman-Keuls and Dunn tests, and the Mann-Whitney rank sum test
where appropriate (significance = P < 0.05).
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RESULTS |
The in vitro isometric contraction studies confirmed
concentration-related stimulation of an increase of phasic contractile activity, consisting of an increase in both frequency and intensity, in
response to PGF2
(Fig.
1A). As
demonstrated in Fig. 1B, the increase
in contractile activity was apparent with 10 nM
PGF2
and statistically
significant at 100 nM and 1 µM compared with strips treated with
vehicle alone (P < 0.05). A
representative recording of simultaneous cytosolic calcium transients
and isometric contractile activity is demonstrated in Fig.
2. As observed,
PGF2
stimulated a small
increase in basal cytosolic calcium along with the development of
calcium oscillations. The cytosolic calcium oscillations were
associated with simultaneous phasic myometrial contractions. Of note,
the relative magnitude of these
PGF2
-stimulated calcium
oscillations and contractions were comparable to the calcium transient
and tonic contraction produced by the brief pulse with 50 mM KCl.

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Fig. 1.
Dose-response effects of prostaglandin
F2
(PGF2 ) on frequency and
intensity of in vitro phasic contractions of a longitudinal strip of
rat uterine tissue. A: representative
tracing of cumulative effects of
PGF2 on isometric contractile
activity, reported in grams of tension generated. Spontaneous
contractions are noted during period preceding 20-min time point;
subsequently, cumulative doses of
PGF2 were added to muscle bath
at 10-min intervals over a concentration range of 1 nM to 1 µM, as
noted. B: cumulative effects of
PGF2 on phasic myometrial
contractions compared with vehicle [dimethyl sulfoxide
(DMSO)] alone. Each bar = mean ± SD;
n = 4-6 experiments. Hatched
bars, strips treated with PGF2 ;
open bars, strips treated with DMSO alone.
* P < 0.05 for bars compared
with vehicle controls.
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Fig. 2.
Representative recording of cytosolic calcium transients and
simultaneous isometric contractile activity of a strip of rat
myometrial tissue loaded with fura 2. Top: changes in relative fluorescent
intensity of intracellular fura 2 after excitation at 340 and 380 nm.
Middle: relative changes in
concentration of cytosolic calcium as indicated by 340-to-380 ratio
(340/380). Bottom: isometric
contractile activity in grams of tension generated in fura 2-loaded
myometrial strip. Myometrial strip was treated with a brief pulse of
KCl (~50 mM) as indicated. Subsequently, infusion of HEPES-buffered
salt solution containing 0.5 µM
PGF2 (as indicated by
horizontal bar) resulted in generation of cytosolic calcium
oscillations and simultaneous phasic contractions.
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NCDC, a membrane-permeant inhibitor of phospholipase C, markedly
suppressed the frequency and intensity of
PGF2
-stimulated in vitro
isometric contractions. As demonstrated in Fig.
3A, the NCDC effect was
reversed after washout and restimulation. This concentration-related
NCDC effect became significant at concentrations of 80 µM and
greater, compared with uterine strips treated with comparable volumes
of vehicle (DMSO) alone (P < 0.05, Fig. 3B). As observed in Fig.
4, NCDC completely inhibited the cytosolic calcium
oscillations and associated contractions produced in response to 0.5 µM PGF2
. Confirming the
important role of the PI-signaling pathway during these phenomena, even
at very low PGF2
concentrations, NCDC completely suppressed cytosolic calcium
oscillations stimulated in response to 10 nM
PGF2
(Fig.
5). As observed in Fig.
6, neomycin, another inhibitor of
phospholipase C, produced significant inhibition of
PGF2
-stimulated phasic
myometrial contractions at concentrations of 1 mM and greater
(P < 0.05). Comparable to NCDC, the
effect of neomycin was reversible with washout of the reagent and
restimulation (Fig. 6A). Consistent
with its effects on phasic contractions, neomycin produced complete
inhibition of PGF2
-stimulated
calcium oscillations (Fig. 7).

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Fig. 3.
Dose-response effects of
2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate
(NCDC) on PGF2 -stimulated
myometrial contractile activity. A:
representative in vitro contraction study demonstrating reversal of
NCDC effect on PGF2 -stimulated
contractions. Activity in grams of tension generated during spontaneous
contraction period preceding 20 min, addition of
PGF2 (1 µM), addition of NCDC
(120 µM), washout with warm saline and fresh buffer, and second
addition of PGF2 .
B: cumulative effects of NCDC on
PGF2 -stimulated (1 µM)
myometrial contractions. Each bar = mean ± SD;
n = 6-8 experiments. Open bars,
strips treated with NCDC; hatched bars, strips treated with vehicle
(DMSO) alone. + P < 0.05 for pretreatment control periods compared with
PGF2 -stimulated contractile
activity; * P < 0.05 for
treatment periods compared with
PGF2 -stimulated contractile
activity and compared with vehicle controls.
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Fig. 4.
Representative recording of cytosolic calcium transients and
simultaneous contractile activity of a fura 2-loaded myometrial strip
treated with 0.5 µM PGF2 and
120 µM NCDC. Top: changes in
relative fluorescent intensity of intracellular fura 2 after excitation
at 340 and 380 nm. Middle: relative
changes in concentration of cytosolic calcium as indicated by 340/380.
Bottom: isometric contractile activity
in grams of tension generated in fura 2-loaded myometrial strip.
Myometrial strip was treated with a brief pulse of KCl (~50 mM) as
indicated. Subsequently, infusion of HEPES-buffered salt solution
containing PGF2 (as indicated
by horizontal bar) resulted in generation of calcium oscillations and
simultaneous contractions; addition of NCDC to infusion resulted in
complete inhibition of these effects.
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Fig. 5.
Representative recording of cytosolic calcium transients and in vitro
contractile activity of a fura 2-loaded myometrial strip treated with
10 nM PGF2 and 120 µM NCDC.
Top: changes in relative fluorescent
intensity of intracellular fura 2 after excitation at 340 and 380 nm.
Middle: relative changes in
concentration of cytosolic calcium as indicated by 340/380.
Bottom: isometric contractile activity
in grams of tension generated. Myometrial strip was treated with a
brief pulse of KCl (~50 mM) as indicated. Subsequently, infusion of
HEPES-buffered salt solution containing this low concentration of
PGF2 (10 nM; as indicated by
horizontal bar) resulted in generation of calcium oscillations and
simultaneous contractions similar to those demonstrated in Fig. 4.
Again, addition of NCDC to infusion resulted in complete inhibition of
these effects.
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Fig. 6.
Dose-response effects of neomycin on
PGF2 -stimulated myometrial
contractile activity. A:
representative in vitro contraction study demonstrating reversal of
neomycin effect, activity in grams tension generated during spontaneous
contraction period preceding 20 min, addition of
PGF2 (1 µM), addition of
neomycin (2.5 mM), washout with warm saline and fresh buffer, and
second addition of PGF2 .
B: cumulative effects of neomycin on
PGF2 -stimulated (1 µM)
myometrial contractions. Each bar = mean ± SD;
n = 6 experiments. Open bars, strips
treated with neomycin; hatched bars, strips treated with vehicle
(distilled water) alone.
+ P < 0.05 for
pretreatment control periods compared with
PGF2 -stimulated contractile
activity; * P < 0.05 for
treatment periods compared with
PGF2 -stimulated contractile
activity and compared with vehicle controls.
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Fig. 7.
Representative recording of cytosolic calcium transients and
simultaneous contractile activity of a fura 2-loaded myometrial strip
treated with 0.5 µM PGF2 and
2.5 mM neomycin. Top: changes in
relative fluorescent intensity of intracellular fura 2 after excitation
at 340 and 380 nm. Middle: relative
changes in concentration of cytosolic calcium as indicated by 340/380.
Bottom: isometric contractile activity
in grams of tension generated in fura 2-loaded myometrial strip.
Myometrial strip was treated with a brief pulse of KCl (~50 mM) as
indicated. Subsequently, infusion of HEPES-buffered salt solution
containing PGF2 (as indicated
by horizontal bar) resulted in generation of calcium oscillations and
simultaneous contractions. Addition of neomycin to infusion resulted in
complete inhibition of these effects.
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As discussed previously, the cycling of intracellular calcium into and
out of the endoplasmic reticulum calcium stores is an important
component of the intracellular mechanisms underlying classic cytosolic
calcium oscillations; the same phenomena appear to be used during
PGF2
-stimulated phasic
myometrial contractions. Modulation of
IP3-dependent calcium release by
thimerosal, a membrane-permeant modulator of
IP3-receptor activity and calcium
uptake, resulted in a biphasic effect on
PGF2
-stimulated phasic
contractions. Lower concentrations of thimerosal mildly stimulated
myometrial contractions; whereas, higher concentrations significantly
inhibited the contractile activity (P < 0.05, Fig.
8B). As
demonstrated in Fig. 8A, reversal of
the effects of this sulfhydryl reagent required washout followed by
treatment of the tissue with dithiothreitol (DTT), a thiol-reducing
agent, and restimulation with
PGF2
. In the absence of the DTT
pretreatment, the second addition of PGF2
was ineffective (data not
shown). As observed in Fig. 8, thimerosal inhibition of phasic
contractions is associated with a transient increase in basal muscle
tension; the cytosolic calcium imaging studies confirmed a comparable
rise in cytosolic calcium as thimerosal inhibited the calcium
oscillations in myometrial tissue (Fig. 9).

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Fig. 8.
Dose-response effects of thimerosal (THIM) on
PGF2 -stimulated myometrial
contractile activity. A:
representative in vitro contraction study demonstrating reversal of
THIM effect, activity in grams tension generated during spontaneous
contraction period preceding 20 min, addition of
PGF2 (1 µM), addition of THIM
(500 µM), washout with warm saline and fresh buffer, addition of 1 mM
dithiothreitol (DTT), a second washout with warm saline and fresh
buffer, followed by a second addition of
PGF2 .
B: cumulative effects of THIM on
PGF2 -stimulated (1 µM)
myometrial contractions. Each bar = mean ± SD;
n = 9 experiments. Open bars, strips
treated with THIM; hatched bars, strips treated with vehicle (distilled
water) alone. + P < 0.05 for pretreatment control periods compared with
PGF2 -stimulated contractile
activity; and * P < 0.05 for
treatment periods compared with
PGF2 -stimulated contractile
activity and compared with vehicle controls.
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Fig. 9.
Representative recording of cytosolic calcium transients and
simultaneous contractile activity of a fura 2-loaded myometrial strip
treated with 0.5 µM PGF2 and
500 µM THIM. Top: changes in
relative fluorescent intensity of intracellular fura 2 after excitation
at 340 and 380 nm. Middle: relative
changes in concentration of cytosolic calcium as indicated by 340/380.
Bottom: isometric contractile activity
in grams of tension generated in fura 2-loaded myometrial strip.
Myometrial strip was treated with a brief pulse of KCl (~50 mM) as
indicated. Subsequently, infusion of HEPES-buffered salt solution
containing PGF2 (as indicated
by horizontal bar) resulted in generation of calcium oscillations and
simultaneous contractions. Addition of THIM to infusion resulted in an
elevation of basal cytosolic calcium concentration and elevation of
basal muscle tension, along with complete inhibition of calcium
oscillations and phasic contractile activity.
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As noted previously, adenine has been reported to inhibit CICR.
Consistent with this reported effect, adenine reversibly inhibited PGF2
-stimulated phasic
contractions, as observed in Fig. 10A. At
concentrations of 1 mM and greater, this adenine effect was significant
compared with uterine strips treated with comparable volumes of vehicle
alone (P < 0.05, Fig.
10B). As demonstrated in Fig.
11, the effects of adenine on contractile
force appeared greater than its effects of calcium oscillations. These
observations suggest that adenine has a potent effect on the
contractile apparatus in uterine myocytes, in addition to its effects
of CICR. Confirming that CICR from the intracellular ryanodine
receptors play an important role during these phenomena, calcium
imaging studies performed using ruthenium red demonstrated suppression
of the frequency and intensity of
PGF2
-stimulated calcium
oscillations and associated contractions (Fig.
12). A previous report from our
laboratory has confirmed the ability of ruthenium red to markedly
inhibit in vitro myometrial contractions stimulated in response to
several uterotonic agonists, including
PGF2
(22).

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Fig. 10.
Dose-response effects of adenine on
PGF2 -stimulated myometrial
contractile activity. A:
representative in vitro contraction study demonstrating reversal of
adenine effect, activity in grams tension generated during spontaneous
contraction period preceding 20 min, addition of
PGF2 (1 µM), addition of
adenine (5 mM), washout with warm saline and fresh buffer, and second
addition of PGF2 .
B: cumulative effects of adenine on
PGF2 -stimulated (1 µM)
myometrial contractions. Each bar = mean ± SD;
n = 6 experiments. Open bars, strips
treated with adenine; hatched bars, strips treated with vehicle
(distilled water) alone.
+ P < 0.05 for
pretreatment control periods compared with
PGF2 -stimulated contractile
activity; and * P < 0.05 for
treatment periods compared with
PGF2 -stimulated contractile
activity and compared with vehicle controls.
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Fig. 11.
Representative recording of cytosolic calcium transients and
simultaneous contractile activity of a fura 2-loaded myometrial strip
treated with 0.5 µM PGF2 and
5 mM adenine. Top: changes in relative
fluorescent intensity of intracellular fura 2 after excitation at 340 and 380 nm. Middle: relative changes
in concentration of cytosolic calcium as indicated by 340/380.
Bottom: isometric contractile activity
in grams of tension generated in fura 2-loaded myometrial strip.
Myometrial strip was treated with a brief pulse of KCl (~50 mM) as
indicated. Subsequently, infusion of HEPES-buffered salt solution
containing PGF2 (as indicated
by horizontal bar) resulted in generation of calcium oscillations and
simultaneous contractions. Addition of adenine to infusion resulted in
marked suppression of calcium oscillations and complete inhibition of
phasic contractions.
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Fig. 12.
Representative recording of cytosolic calcium transients and
simultaneous contractile activity of a fura 2-loaded myometrial strip
treated with 0.5 µM PGF2 and
30 µM ruthenium red (RRED). Top:
changes in relative fluorescent intensity of intracellular fura 2 after
excitation at 340 and 380 nm. Middle:
relative changes in concentration of cytosolic calcium as indicated by
340/380. Bottom: isometric contractile
activity in grams of tension generated in fura 2-loaded myometrial
strip. Myometrial strip was treated with a brief pulse of KCl (~50
mM) as indicated. Subsequently, infusion of HEPES-buffered salt
solution containing PGF2 (as
indicated by horizontal bar) resulted in generation of calcium
oscillations and simultaneous contractions. Addition of RRED to
infusion resulted in marked suppression of frequency and duration of
calcium oscillations and phasic contractions.
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During classic cytosolic calcium oscillations, the influx of
extracellular calcium appears to be essential for both supplementation of the cytosolic calcium transients and refill of the intracellular calcium stores. A similar need for extracellular calcium appears to
occur during PGF2
-stimulated
phasic myometrial contractions. Consistent with this requirement for
the influx of extracellular calcium, the studies performed using
nifedipine, a membrane calcium channel blocker, confirmed significant
inhibition of PGF2
-stimulated phasic myometrial contractions (P < 0.05, Table 1). Significant inhibition of
PGF2
-stimulated contractile
activity was also observed in response to removal of extracellular
calcium by replacement of the EBSS buffer with calcium-free EBSS buffer
containing 2 mM EGTA (P < 0.05, Table 1).
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DISCUSSION |
The studies described in this report have provided evidence to support
the hypothesis that
PGF2
-stimulated contractions occur in response to the generation of cytosolic calcium
oscillation-like phenomena. The in vitro contraction studies, which
confirmed suppression of
PGF2
-stimulated phasic
myometrial contractions in response to several inhibitors of the
PI-signaling pathway, are quite consistent with the previously reported
effects of these reagents. Of interest, these inhibitors suppressed
phasic contractile activity below baseline activity, suggesting that
the spontaneous activity of myometrial smooth muscle is dependent on
some level of constitutive activity of the PI-signaling pathway. The
cytosolic calcium imaging studies described in this report have clearly
demonstrated periodic calcium transients associated with simultaneous
myometrial contractions in response to
PGF2
; these myometrial calcium
transients are consistent with cytosolic calcium oscillations observed
in other cells. Similar cytosolic calcium oscillation-like phenomena have been reported in response to oxytocin and other uterotonic agonists using both cultured uterine myocytes and strips of myometrial tissue (3, 8, 12, 23, 24, 28).
The cytosolic calcium results described in our studies using fura 2, a
ratiometric calcium indicator, have been reported in relative changes
in cytosolic calcium based on 340/380; however, other investigators
have reported the absolute concentrations of cytosolic calcium in
myometrial tissue. Szal et al. (28) used aequorin- (a calcium-sensitive
bioluminescent protein) loaded human myometrial tissue to measure
resting cytosolic calcium levels of 158 nM and peak stimulated levels
of 210-390 nM. These investigators also observed that the
half-maximal effective calcium concentration with respect to force
generation was 172 nM in myometrial tissue. Anwer et al. (1) and
Criswell et al. (3), using fura 2, a ratiometric calcium indicator,
have reported similar basal and stimulated cytosolic calcium
concentrations in cultured uterine myocytes. Because of concerns
regarding the potential difficulty in exactly reproducing the
intracellular molecular conditions during the calibration of these
calcium indicator dyes, many investigators are currently reporting
cytosolic calcium data in relative changes based on 340/380 (e.g.,
Refs. 8 and 12 and as done in our study) rather than absolute
concentrations of cytosolic calcium.
Until recently, the signal transduction mechanisms mediating the
intracellular effects of prostaglandins, especially PGE and PGF2
, have been unclear. Using
corpus luteal cells, Leung et al. (10) demonstrated in 1986 that
PGF2
stimulated a rapid,
concentration-related decrease in membrane PI mono- and bisphosphates,
along with an increase in inositol polyphosphates, including
IP3. Similar phenomena have been
reported in myometrial tissue in response to stimulation with PGs,
especially PGF2
. Using cultured
human myometrial cells, Molnár and Hertelendy (16) reported an
increase in inositol phosphate production in response to 1-10 µM
PGF2
, albeit to a lesser degree
than that produced by oxytocin. However, these investigators have
suggested that the effects of
PGF2
, especially at low
nanomolar concentrations, are dependent on the influx of extracellular
calcium by mechanisms unrelated to activation of phospholipase C and
the generation of IP3 (15, 17). In
contrast, other investigators have clearly observed a relationship
between PGF2
stimulation,
activation of the PI-signaling pathway, and elevation of cytosolic
calcium. In 1992, Goureau et al. (5) demonstrated the rapid,
concentration-related stimulation of inositol phosphate production in
both estrogen-dominated and pregnant rat myometrial tissue in response
to PGs; PGF2
was found to be
the most potent of the prostanoids tested. More recently, Phaneuf et
al. (20) reported PGF2
activation of the PI-signaling pathway in cultured human myometrial
cells. In contrast to the reports by Molnár and Hertelendy (15,
17), these investigators found that the effects of
PGF2
were not dependent on the
influx of extracellular calcium. Phaneuf et al. (20) have also provided
evidence demonstrating that members of the
Gq class of G protein
-subunits
couple membrane PGF receptors to the PI-signaling pathway in uterine
myocytes. Our observations that
PGF2
-stimulated calcium
oscillations and phasic contractions were markedly suppressed by
membrane permeant phospholipase C inhibitors provide further evidence
that the PI-signaling pathway plays an important role during these
phenomena. Interestingly, these inhibitor effects were apparent even at
low nanomolar concentrations of
PGF2
. Addressing the
facilitative role of extracellular calcium during these signaling
phenomena, Maka et al. (13) observed that extracellular calcium
depletion completely inhibited inositol phosphate production stimulated
in response to both oxytocin and PGF2
. Therefore, extracellular
calcium appears necessary, not only for the supplementation of the
cytosolic calcium transient and refill of the intracellular calcium
stores, but also to facilitate the activation of agonist-stimulated
phospholipase C in myometrial smooth muscle cells.
Recently the PG membrane receptors have been characterized at a
molecular level. PGE receptors have been reported to activate multiple
second messenger pathways, including modulation of adenylate cyclase
activity, inositol phosphate production, and regulation of cytosolic
calcium (18). In contrast, the PGF receptor appears to be coupled to a
second messenger pathway, resulting only in the activation of the
PI-signaling pathway and stimulation of increased cytosolic calcium
(27). To date, four distinct PGE receptors have been cloned and
sequenced; each was found to be coupled to unique intracellular
signaling pathways (18). In contrast, only one PGF receptor has been
cloned and sequenced; the PGF receptor contains 366 amino acids,
resulting in a calculated molecular weight of 40 kDa (27). The
secondary structure of the PGF receptor is consistent with that of
other G protein-coupled receptors, and transfection of COS cells with
cDNA for this receptor resulted in
PGF2
-stimulated activation of
the PI-signaling pathway (27). The data described in the present report
have confirmed that comparable PGF receptor-coupled signaling
mechanisms are used during
PGF2
-stimulation of
phasic myometrial contractions.
Perspectives.
To date, the intracellular mechanisms underlying
PGF2
-stimulated phasic
myometrial contractions have not been well defined. The studies
described in our report continue to address this situation. Our studies
have provided evidence supporting the hypothesis that PGF2
stimulation of the
PI-signaling pathway results in the generation of cytosolic calcium
oscillation-like phenomena. These intermittent calcium transients,
arising from both the release of intracellular calcium and the influx
of extracellular calcium, appear to result in the activation of the
contractile proteins and the generation of phasic myometrial
contractions. We have reported similar signal transduction phenomena
occurring in response to other uterotonic agonists, including oxytocin,
aluminum fluoride (a direct G protein agonist), moderate elevation of
extracellular potassium (10-30 mM), ionomycin (a calcium
ionophore), BAY K 8644 (a calcium channel agonist), and others
(21-24). All of these observations suggest that there is a common
intracellular signaling pathway occurring in uterine myocytes that
results in the consistent generation of phasic contractile activity in
response to uterotonic agonists. With an understanding of these
intracellular mechanisms, future studies can begin to explore the
hormonal and physiological mechanisms that regulate the expression and
activation of this myometrial signaling pathway. Such knowledge is
essential to understand and better pharmacologically treat medically
important disturbances of myometrial activity, especially preterm labor
and premature delivery.
 |
ACKNOWLEDGEMENTS |
This research was funded by National Institute of Child Health and
Human Development research Grants HD-22063 and HD-28506.
 |
FOOTNOTES |
Address for reprint requests: M. Phillippe, Dept. of Obstetrics and
Gynecology (MC2050), Univ. of Chicago, 5841 S. Maryland Ave., Chicago,
IL 60637.
Received 5 November 1996; accepted in final form 20 June 1997.
 |
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