BIOLOGY OF REPRODUCTION 52, 1244-1249 (1995)
Effect of Purinergic Stimulation on Intracellular Calcium Concentration and
Transepithelial Potential Difference in Cultured Bovine Oviduct Cells'
CHRISTOPHER I. COX 2 and HENRY J. LEESE
Department of Biology, University of York, Heslington, York, United Kingdom
ABSTRACT
Epithelial cells were removed from bovine oviducts by enzyme digestion and either cultured on laminin-coated coverslips
(for determination of [Ca2+]i) or on collagen filters (for determination of transepithelial potential difference [pd]). Cells on cover2
2
slips were loaded with Fura-2 to monitor [Ca +]. Application of extracellular ATP induced a transient increase in [Ca +]i in a
2
dose-dependent manner. This response was abolished by thapsigargin, indicating that the rise in [Ca +]i was derived from intra2
cellular stores. The order of potency of the nucleotide-induced rise in [Ca +]i was uridine triphosphate (UTP)>ATP>ADP. Epithelial cells were grown on collagen filters, and when mounted in a modified Ussing chamber exhibited an electrical pd of 1.00
± 0.36 mV with the apical side negative with respect to the basal. Application of UTP, ATP, and ADP to the basal side induced
transient increases in pd of 1.15 + 0.21, 0.77 + 0.16, and 0.26 ± 0.06 mV, respectively. The order of potency of the nucleotides
2
in eliciting transient increases in [Ca +]i and pd suggests the presence of a P2, purinergic receptor in the bovine oviduct epithelium that could play a role in transepithelial ion movements and hence the control of oviductal fluid formation.
INTRODUCTION
The oviduct is the site of gamete transport, fertilization,
and early embryo development. The epithelial cells lining
the inner surface of the oviduct are ultimately responsible
for the formation of the oviduct fluid, which presumably
provides the optimum environment for these reproductive
processes [1]. Although information is available on the composition of oviduct fluid in a number of species [2-4], little
is known about its mode of formation.
Using a combined vascular and luminal perfusion technique in the rabbit oviduct, Gott et al. [5] found that oviduct
fluid formation was linked to the secretion of chloride ions
(Cl-). Dickens et al. [6] subsequently reported a net flux of
C1- in the secretory direction across a monolayer of rabbit
oviduct cells mounted in an Ussing chamber, in confirmation of an earlier report by Brunton and Brinster [7].
Dickens and Leese [8] then showed that rabbit oviduct fluid
formation during vascular perfusion is completely inhibited
by dibutyryl cyclic AMP (db cAMP) applied to the vascular
compartment and is also responsive to adrenergic agonists
and antagonists such as isoprenaline and propranolol. These
reports are among the very few to have addressed the
mechanisms or control of oviduct fluid formation.
Extracellular ATP has been demonstrated to regulate a
variety of biological processes (for reviews see [9] and [10]).
Such effects are mediated through specific purinergic receptors and include the control of transepithelial ion transport. For example, purinergic receptors have been reported to regulate transepithelial ion transport in airway
Accepted January 23, 1995.
Received July 25, 1994.
'This work was supported by the European Union Biotechnology Grant B102CT92-0163.
2
Correspondence. Department of Biology, University of York, Heslington, York,
Y01 5DD, UK. FAX: (0904) 432860.
[11, 12], epididymal, [13], renal [14, 15], and intestinal [16,17]
epithelia. The response to ATP appears to be transduced by
an increase in intracellular calcium concentration ([Ca2+]i),
which activates a variety of secondary biochemical reactions such as stimulation of Ca2+-calmodulin kinase and by
direct activation of membrane ion channels (e.g., Ca 2+ activated K+ channels). Several classes of P2 purinergic receptors exist, characterized by their agonist rank order of
potency. However, there are no reports of the presence of
purinergic receptors in the oviduct epithelia of any species.
We have used two methods to study the effect of purinergic agonists on epithelial cells from the cattle oviduct.
In the first, cells isolated by enzyme digestion have been
plated onto laminin-coated glass coverslips for measurement of [Ca 2+]i using the Ca2+-sensitive fluorochrome Fura2. In the second, epithelial cells have been grown in primary culture on permeable collagen filters. When cultured
in this manner, oviduct and uterine epithelial cells exhibit
a polarized morphology, as is the case in situ [6,18, 19].
This method has been employed to study the effect of purinergic agonists on the electrical potential difference (pd)
across the polarized epithelial monolayers.
A preliminary account of some of this work has been
presented to the Society for the Study of Fertility [20].
MATERIALS AND METHODS
Cell Preparation
Epithelial cells were isolated according to the method
of Dickens et al. [6], which is similar to that used by Kimber
et al. [19] for the isolation of mouse uterine epithelia, which
was, in turn, derived from that of Glasser et al. [18]. Oviducts were obtained from a local abattoir from heifers judged
from the appearance of the ovaries to have ovulated in the
past three days [21]. The oviducts were immediately washed
1244
CALCIUM-MEDIATED PURINERGIC RECEPTOR IN BOVINE OVIDUCT EPITHELIA
in ice-cold Ca 2+ - and Mg2+-free Hanks' buffered salt solution (HBSS) containing penicillin (270 U ml-'), streptomycin (270 mg ml-'), Fungizone (20 mg ml-') (all purchased from Gibco Life Technologies Ltd., Paisley, UK), and
nystatin (1 mg ml-l; Sigma Chemical Co., Poole, Dorset,
UK). They were opened longitudinally, cut into approximately 10-mm lengths, placed in 20 ml enzyme medium
(0.5% trypsin [type III, Sigma] and 2.7% pancreatin [Gibco]
in HBSS), and kept on ice for 1 h and then at ambient temperature for another 1 h. The enzyme medium was removed from the oviduct pieces, 10 ml ice-cold HBSS was
added, and the mixture was vortexed for 10 sec. The cloudy
supernatant was collected, another 10 ml HBSS was added,
and the mixture was vortexed again. The supernatants were
combined and centrifuged at 2000 rpm for 5 min in an MSE
Centaur 2 bench top centrifuge (Fisons Loughborough, UK).
The resulting pellet was washed three times by resuspension and centrifugation in HBSS and resuspended in culture medium (Nutrient mixture F-12; Sigma) plus DMEM
(1:1 v/v) containing 0.1% BSA (ICN-Flow, Oxfordshire, UK),
270 U ml- penicillin, 270 mg ml - ' streptomycin, 20 mg
ml-' Fungizone, 1 pIg ml l nystatin, 2.5 mM glutamine
(Sigma), 2.5% Nu-serum (ICN-Flow), and 2.5% heat-inactivated fetal bovine serum (Gibco) at a concentration of 5 x
104 cells ml-'. Cell viability, assessed by the cell's ability to
exclude 0.4% w/v trypan blue dye (Sigma), was found to
be > 95% in all preparations. For determination of pd, 0.25
ml of cell suspension (1.25 x 10 4 cells) was placed on 14mm diameter collagen filters (ICN-Flow) in the center 8
wells of 24-well tissue culture plates (Falcon; A & J Beveridge Ltd., Newcastle-upon-Tyne, UK). Fresh medium (0.45
ml) was placed on the underside of the filters. For Fura-2
studies, 0.1 ml cell suspension was plated onto 10-mm diameter glass coverslips pretreated with laminin (Sigma) at
1 [Lg cm-2. For immunocytochemical staining, cells were
seeded onto multiwell slides (Falcon) at a density of 5 x
103 cells per well. All cell preparations were maintained at
37°C in 5% CO2:95% air and fed with fresh medium every
24 h.
Immunocytochemistry
After 5 days in culture, the cells on multiwell slides were
washed twice with PBS prior to being fixed for 2 min in
methanol/acetone (1:1 v/v). The slides were allowed to airdry. Twenty microliters of the LP1K primary antibody against
cytokeratin 8 was added, and the cells were incubated for
1 h at ambient temperature in a humidified box. The slides
were washed three times in PBS, fixed again in methanol/
acetone, and allowed to air-dry. Ten microliters of a fluorescein isothiocyanate (FITC)-labeled secondary antibody
was added, and incubation was continued for 30 min at
ambient temperature in the humidified box. The slides were
washed three times in PBS containing 0.25% Tween 20
(Sigma) and three times with distilled water, air-dried, and
examined under an epifluorescence microscope.
1245
[Ca2+]iMeasurement
Cells on laminin-coated coverslips were incubated in
media containing 10 mM Fura-2 AM (Calbiochem, Nottingham, UK) for 60 min at 37 0C in 5% CO 2:95% air. The
coverslips were mounted in a small perfusion chamber on
the stage of a Nikon Diaphot EPI fluorescent microscope
fitted with an excitation filter changer and photon counting
system [22] (Newcastle Photometric Systems Ltd., Newcastle-upon-Tyne, UK). Single cells were visualized and were
excited alternately with light at 350 and 380 nm. The fluorescence emitted at wavelengths > 520 nm was recorded,
and ratio measurements were made every 1.1 sec. Solutions
preheated to 37°C were perfused through the experimental
chamber at a rate of 3 ml/min-' for up to 1 h. The normal
perfusion solution contained 94.7 mM NaCl, 4.78 mM KCI,
1.19 mM KH 2PO 4 , 1.19 mM MgSO 4, 23.28 mM sodium lactate, 5.56 mM glucose, 0.33 mM sodium pyruvate, 1.71 mM
CaC 2, 4.0 mm NaHCO 3 , and 19.5 mM HEPES (pH 7.4). A
Ca2+-free solution was prepared by omitting Ca 2+ and adding 0.1 mM EGTA. Calibration of the system used solutions
of Ca2+/EGTA complex of known free Ca2 + concentration
(Molecular Probes Inc., Eugene, OR) with known concentrations of the pentasodium salt of Fura-2 (Calbiochem). This
method of calibration, although prone to error, does provide an estimate of [Ca 2 +]i [23].
Electricalpd Measurement
Epithelial cells on collagen filters were clamped between the two halves of a modified perspex Ussing chamber maintained at 37 0C. Two milliliters of the perfusion medium described above was added gradually to each side of
the filter to avoid forming pressure gradients across the cell
monolayer. Both sides of the filter were gassed with 5%
CO2 :95% 02 to ensure adequate oxygenation and mixing.
Agar bridges were inserted into each side of the chamber
and linked, via 3 M KCl, to matched calomel electrodes
connected to a Vibron Electrometer. The pd was continuously displayed on a flat-bed chart recorder. Nucleotides
were added to the basal side of the filters in a volume of
0.1% of the total, to minimize dilution and pressure gradient effects.
RESULTS
The LP1K antibody to cytokeratin 8 reacted intensely with
the cultured epithelial cells. More than 95% of the cells
isolated stained positively, providing strong evidence that
a homogeneous epithelial cell population had been isolated (Fig. 1).
Epithelial cells grown on laminin-coated coverslips were
incubated for 1 h with the ester form of the calcium-sensitive fluorochrome Fura-2. The cells emitted a strong fluorescence signal at > 520 nm when excited at 350 and 380
nm, indicating that the dye had been taken up and cleaved
to the unesterified form. Extracellular ATP, perfused over
1246
COX AND LEESE
a4.0
TV v
T
300-
eq
2004
U
U
100-
0
.
-9
-8
.
-7
.·
.
-6
.
.
-5
.....·
-4
.
i
-3
-2
log ATP (M)
FIG. 3. Dose-response curve of ATP on transient increase above basal
levels in [Ca2+lJ in single bovine oviduct epithelial cells loaded with Fura2. Each point represents mean + SE (n = 8).
FIG. 1. Immunohistochemical labeling of epithelial specific cytokeratin
in primary cultures of bovine oviduct cells. Immunofluorescent staining was
performed as described in Materials and Methods with use of LP1K antibody against cytokeratin 8. Bar = 20 m.
2.0
1.5
o
r.
4,
ae
1.0
0.5
III
0
100
200
Time (sec)
FIG. 2. Original trace of [Ca2 +11from Fura-2-loaded single bovine oviduct epithelial cell exposed to ATP. The ICa2 ]i response to extracellular
ATP is manifested as a single transient Ca2+ spike. Upper line represents
2
duration of exposure to 10 IM ATP. Ca+],
was estimated as described in
Materials and Methods. Trace is representative of 30 such experiments.
the cells, gave rise to a pronounced transient increase in
[Ca2 +]i (Fig. 2). Calibration with EGTA/Ca 2+ buffers of known
free Ca 2+ concentration indicated that [Ca2+Ji rose from a
resting value of about 80 nM to a peak of approximately
350 nM. The duration of the elevation above basal levels
was about 30 sec, and the rise and fall of the peak was
symmetrical. The response to extracellular ATP was shown
to be dose-dependent; the minimum concentration that gave
a detectable increase was 10 nM, and the response plateaued at 100 p.M (Fig. 3).
Thapsigargin may be used to deplete intracellular calcium stores irreversibly [24]. The trace shown in Figure 4
demonstrates that preincubation with 20 ,pM thapsigargin
totally abolished the calcium spike associated with the response to extracellular ATP.
In order to characterize the purinergic receptor in more
detail, the effect of a series of nucleotides on the agonistinduced increase in [Ca 2+]i was examined. The order of potency of those nucleotides, tested at a concentration of 100
RIM, was uridine triphosphate (UTP)>ATP>ADP (Fig. 5).
Bovine oviduct epithelial cells grown on the permeable
collagen supports mounted in a modified Ussing chamber
exhibited a small but stable electrical pd of 1.0 ± 0.36 mV
(n = 26) with the basal compartment positive with respect
to the apical. Application of 100 pM ATP to the basal compartment significantly increased (p < 0.01) the pd by 0.77
+ 0.16 mV (n = 6) (Fig. 6). UTP and ADP added to a final
concentration of 100 [LM also increased the transepithelial
pd, with the order of potency similar to that shown for the
rise in [Ca2+], i.e., UTP>ATP>ADP (Fig. 7).
DISCUSSION
We have shown that bovine oviduct epithelial cells in
primary culture exhibit transient increases in [Ca2+]i in re-
1247
CALCIUM-MEDIATED PURINERGIC RECEPTOR IN BOVINE OVIDUCT EPITHELIA
2.0.
1
Thapsigargin
i
r
.
ATP
.
.
.
I
.
'300
o
W.
I.
1.5
'200
co
+
1'l
0
g
I
el
'I,,
,100
1.0_ y.
U""~~-·5.·,
",Z*
. .........
r
.......
(>
50
0.5w
U
100
0
400
300
200
500
Time (sec)
FIG. 4. Effect of 20 1iM thapsigargin and subsequent 10 ~tM ATP stimulation on ICa2+ij in Fura-2-loaded single bovine oviduct epithelial cells. Upper
line represents duration of application of thapsigargin and ATP. Trace is representative of 10 such experiments.
sponse to extracellular nucleotides, notably, ATP. To the best
of our knowledge, this is the first report on the existence
of purinergic receptors in the mucosal lining of the mammalian female reproductive tract. By analogy with other
transporting epithelia, this response is likely to be involved
in the regulation of oviduct fluid secretion. The data presented are for oviducts removed from animals in the pe-
2.5-
ATP
pd (mV)
2.0-
400
-T1
0
300-
1.5-
a
.I
U
-
+
.
0
A
w
Q
200
1.0-
100-
I
I
0
0ADP
FIG.
oviduct
plied to
mean
ATP
5. Elevation in [Ca2+] induced by nucleotides in single bovine
epithelial cells loaded with Fura-2. ADP, ATP, and UTP were all apepithelial cells at concentration of 100 tIM. Each column represents
SE (n = 9).
--
I
2
I
4
!
6
Time (min)
8
8
FIG. 6. Original chart recording of transepithelial pd following basal
application of 100 FzM ATP. Transepithelial pd of cells grown on collagen
filters was carried out as described in Materials and Methods. Trace is representative of 9 such experiments.
1248
COX AND LEESE
1.4-
T
1.2E
1.0-
'
.9
0.8-
C
0.6-
tq
0.4-
-T-
0.2 0.0 -
--
ADP
ATP
UIP
FIG. 7. Elevation of electrical potential difference across polarized
monolayers of bovine oviduct epithelia on collagen filters in response to
nucleotides applied at concentration of 100 M to basal compartment. Each
column represents mean + SE (n = 9).
riod 3 days postovulation. Preliminary data for cells isolated
at other stages indicate that the response is independent of
the stage of the reproductive cycle, but more work is required to confirm this.
Bovine oviduct epithelial cells were isolated by enzyme
digestion as a virtually pure cell population as shown by
immunocytochemical characterization using an antibody
raised against the epithelial-specific cytokeratin 8. Cells were
then cultured in two ways: on laminin-coated glass coverslips and on collagen filters. The cells on coverslips formed
a single layer that could be used for measurements of [Ca 2+],.
Both preparations were viable for up to 2 wk. When grown
on permeable collagen supports, the cells formed a confluent monolayer in 5-7 days. The monolayers exhibited
asymmetry in glucose uptake and lactate formation, which
were greater at the basal than apical pole of the cells (Cox
and Leese, unpublished). This observation is similar to that
reported for rabbit oviduct epithelial cells [25].
When incubated with the cell-permeant dye Fura-2 AM,
a fluorescence signal ratio could be detected, corresponding to an estimated intracellular free calcium concentration
of approximately 80 nM. The signal was stable for up to 1
h. Application of ATP resulted in a transient increase in [Ca2+]i
(Fig. 2) in a dose-dependent manner. The properties of the
receptor were characterized by testing the effect of the nucleotides UTP and ADP. The observed order of potency
(UTP>ATP>ADP) in inducing a rise in [Ca2 +],, corresponds
to the presence of the P2 u subclass of purinergic receptors
[10].
Stimulation of the cells with ATP either in the presence
of caffeine or in the absence of extracellular Ca2+ initially
suggested that the rise in [Ca2+]j was dependent on both
intracellular and extracellular sources of Ca 2+ [20]. However, when thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca 2+-ATPase, was used to empty the in-
tracellular Ca2 + stores, it was observed that the response to
ATP was totally abolished. In the absence of extracellular
Ca2+ it is probable that the cytosolic free Ca 2+ is reduced
and the intracellular stores are thus reduced in order to
compensate for the loss in [Ca 2+]i. The observation that the
ATP-induced rise in [Ca 2+ ]i is solely derived from intracellular stores is consistent with the P2, class of purinergic
receptor [10].
The low electrical pd observed across the monolayers
of bovine oviduct epithelial cells, mounted in a modified
Ussing chamber, indicates that the cells form a low-resistance epithelium in primary culture as is thought to be the
case in situ [1]. The transient increase in transepithelial pd
in response to basal application of ATP, UTP, and ADP
strongly suggests a functional role for nucleotides in the
control of oviductal fluid formation. ATP has been shown
to influence the formation and composition of secretions
formed by epithelia from a number of different tissues
[13, 16, 26, 271. In these epithelia, the most widely characterized ion movement in response to ATP addition is of Cl-.
It is therefore of interest that Gott et al. [5] reported that
the rabbit oviduct was lined by a C1--secreting epithelium.
However, it is also possible that the transepithelial movements of other ions such as Na+ , K+ , and Ca2+ are involved
in the increase of the transepithelial pd in response to nucleotides. Oviduct fluid is characterized by a high concentration of K+ and to a lesser extent Ca2 + , in comparison
with serum [1]; indeed, James and Okada [28] have reported the presence of a Ca2+-activated K+ channel in the
apical membrane of rabbit oviduct epithelial cells.
Other reports pertaining to the control of oviduct fluid
formation are concerned with adrenergic stimulation [6, 29],
where ot and
adrenergic agonists have been demonstrated to produce a transient increase in pd similar to that
observed for ATP. Dickens et al. [6] also reported a basalto-apical movement of Cl- consistent with the model proposed by Gott et al. [5].
In conclusion, we have shown that bovine oviduct epithelial cells may readily be isolated and grown in culture
in a manner that permits the ionic and electrophysiological
properties of polarized monolayers to be studied in detail.
This approach will also allow the role of Ca 2+ , and other
intracellular messengers, in modulating oviduct fluid secretion to be characterized at the single cell level.
NOTE ADDED IN PROOF
Since submitting this paper, Dickens and Leese 0 Reprod Fertil 1994; 13:47 (abstract) and Squires et al Physiol 1995; 482: 45P (abstract) have reported the presence of purinergic receptors in human fallopian tubal epithelial cells.
ACKNOWLEDGMENTS
The authors thank Meg Stark for carrying out the transmission electron microscopy. We would also like to thank Claire Dickens for helpful discussion.
CALCIUM-MEDIATED PURINERGIC RECEPTOR IN BOVINE OVIDUCT EPITHELIA
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