Department of Obstetrics and Gynecology, University MacDonald Women's Hospital, University Hospitals of Cleveland, and Departments of Reproductive Biology and Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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ABSTRACT |
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Micromolar concentrations of
ATP stimulate biphasic change in transepithelial conductance across
CaSki cultures, an acute increase (phase I response) followed by a
slower decrease (phase II response). Phase I and
phase II responses involve two distinct calcium-dependent
pathways, calcium mobilization and calcium influx. To test the
hypothesis that phase I and phase II responses
are mediated by distinct P2 purinergic receptors, changes in
permeability were uncoupled by blocking calcium mobilization with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA) or by lowering extracellular calcium, respectively. Under these
conditions ATP EC50 was 25 µM for phase I
response and 2 µM for phase II response. The respective
agonist profiles were ATP > UTP > adenosine
5'-O-(3-thiotriphosphate) (ATP-S) = N6-([6-aminohexyl]carbamoylmethyl)adenosine
5'-triphosphate (A8889) > GTP and UTP > ATP > GTP = A8889 > ATP-
S. Suramin blocked phase I
response and ATP-induced calcium mobilization, whereas pyridoxal phosphate-6-azophenyl-2',4-disulfonic acid (PPADS) blocked phase II response and ATP-augmented calcium influx. ATP time course and
pharmacological profiles for phase II response and augmented calcium influx were similar, with a time constant of 2 min and a
saturable concentration-dependent effect (EC50 of 2-3
µM). RT-PCR experiments revealed expression of mRNA for both the
P2Y2 and P2X4 receptors. These results suggest
that the ATP-induced phase I and phase II
responses are mediated by distinct P2 purinergic receptor mechanisms.
cervix; epithelium; paracellular permeability; transport
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INTRODUCTION |
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EPITHELIAL CELLS of the uterine cervix regulate secretion of fluid that lubricates the cervical and vaginal canals. The product of transcervical transport, cervical plasma, is important for human reproduction and health. It originates by transudation of fluid and solutes from the blood into the cervical canal and is driven by the transepithelial hydrostatic gradient between blood and lumen (9).
Two types of secretory epithelia line the uterine cervix, the
monolayered endocervical epithelium and the stratified ectocervical epithelium. Cervical epithelia, like other types of secretory epithelia, are organized as layers of confluent cells, in which plasma
membranes of neighboring cells come into close contact and functionally
occlude the intercellular space. Molecules can move across
epithelia either through the cells (transcellular route) or via the
intercellular space (paracellular route). Human cervical cells form
relatively leaky types of epithelia, and their overall permeability
properties are determined by the paracellular route. For example, CaSki
cells, a model of endocervical cells (18), form confluent
and polarized epithelium on filters, with baseline levels of
transepithelial electrical conductance (GTE) of
~100 mS/cm (10 · cm2). In CaSki epithelium,
as well as in other cultured epithelia derived from human cervical
epithelial cells, the solute permeability for molecules that traverse
the epithelium via the paracellular route ranges from 0.5 to 20 × 10
6 cm/s in the molecular mass range of 0.18-70 kDa
(18), indicating that human cervical epithelial cells form
a relatively permeable (leaky) epithelium on filters.
Fluid and solute movement via the paracellular route is determined by the resistance of the lateral intercellular space (RLIS) and by the resistance of the intercellular tight junctions (RTJ) in series (18, 27, 31). RLIS is considered a low resistive element and is determined by the proximity of plasma membranes of neighboring cells and the length of the intercellular space from tight junctions to basal lamina. In contrast, the regions of tight junctions are considered a high resistive element, because of the occlusion of the intercellular space by the tight junctional complexes. In cultured human cervical epithelia RLIS and RTJ can be regulated independently and assayed separately (14, 17, 18). Previous studies in human cervical cells showed that micromolar concentrations of ATP stimulate acute changes in RLIS and RTJ (12, 14, 17), making these cultures an important system to study regulation of paracellular permeability by P2 receptors.
ATP elicits biphasic change in paracellular permeability, an acute increase followed by a slower and sustained decrease in permeability (12). This effect can be described in terms of activating ATP receptor(s) located on the apical (luminal) cell surface (12, 14, 15) and is specific to cervical cells. Similar responses were obtained in a number of different types of human cervical epithelial cells including normal ectocervical and endocervical cells, HT3 and ECE16-1, but not in other types of cultured epithelia such as human keratinocytes, human intestinal HT-29 Cl cells, or rabbit proximal tubule cells (16).
Effects triggered by extracellular ATP were first reported by Drury and Szent-Gyorgyi (5) in 1929 based on the ability of ATP as a peripheral neurotransmitter to contract smooth muscle. Receptors activated by ATP have since been characterized and designated purinergic receptors (21, 26) and classified into P2Y and P2X receptors (14, 21, 26). In the female reproductive tract ATP, acting through purinergic receptors, is utilized as a nonadrenergic, noncholinergic cotransmitter to smooth muscle and is also released from nonneuronal cells to act on P2 receptors in the myometrium (8, 23) and fallopian tube (30). Extracellular ATP, acting via P2Y receptors, stimulates increases in the prostaglandin-synthesizing capacity of endometrium and myometrium (1); it regulates human fallopian tube fluid formation (4) and is a potent regulator of transepithelial transport of solutes and fluid across the human cervical epithelium (12, 14, 16, 17, 31). A P2 receptor has been identified by solubilization of human uterine membranes (28). Human cervical cells express P2Y2 receptor (11), and rat uterine tissues express P2X receptors (2, 29).
In human cervical epithelial cells the responses to ATP and the paracellular effectors involved were recently described (11, 12, 14, 17). On the basis of these studies we tested the hypothesis that the effects of ATP are mediated by activation of two distinct P2 receptor mechanisms. To further test this hypothesis, studies were conducted using P2 receptor agonists and antagonists under experimental conditions that uncouple phase I and phase II responses by uncoupling calcium signaling.
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METHODS |
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Cell culture techniques. CaSki cells, which retain phenotypic characteristics of human endocervical cells (18), were grown and maintained in regular medium enriched with 8% fetal calf serum as described previously (18). For most experiments, cells were plated and grown on filters (18) and drugs were added to the luminal and subluminal solutions.
Determinations of changes in cytosolic calcium in cells attached on filters. Changes in cytosolic calcium were determined in cells on filters as described previously (20). Briefly, cells on filters were loaded with 7 µM fura 2 and measurements of fluorescence were conducted in a custom-designed fluorescence chamber.
Determinations of changes in cytosolic calcium in dispersed cells. Changes in cytosolic calcium were determined in dispersed cells as described previously (19). Briefly, cells were plated on Millicel-CM filters for 4-5 days, harvested, and loaded with 1 µM fura 2. Cuvettes containing cells (106 cells/1.5 ml medium) were used in a filter fluorimeter, and changes in cytosolic calcium were determined as described previously (19).
Measurements of GTE.
Measurements of GTE, including calibrations,
controls, and conditions for optimal determinations of
GTE across low-resistance epithelia, were
performed as described previously (17). Briefly, changes
in GTE were determined in Ussing chambers from
successive measurements of the transepithelial electrical current
(I, obtained by measuring the current necessary to clamp
the offset potential to zero and normalized to the 0.6-cm2
surface area of the filter) and transepithelial potential difference (
PD; lumen negative) as GTE =
I/
PD. In CaSki cells determinations of
GTE correlate well with changes in fluxes across
the paracellular pathway (18).
45Ca2+ influx. Cells on filters were shifted for 1 min at 37°C to modified Ringer buffer composed of (in mM) 120 NaCl, 5 KCl, 10 NaHCO3 (before saturating with 95% O2-5% CO2), 1.2 CaCl2, 1 MgSO4, 5 glucose, and 10 HEPES, pH 7.4, with 0.1% bovine serum albumin in volumes of 4.7-5.2 ml in the luminal and subluminal compartments. For experiments, fresh buffer supplemented with 45CaCl (2 µCi/ml) was added for 3 min of preincubation at 37°C. Agonists were added to the solution from concentrated (1,000×) stocks to the luminal and subluminal compartments. At the end of incubation, the medium was removed, and the filter was dipped three times in 3 liters of ice-cold Ca2+-free buffer to remove extracellular isotope. Fresh buffer (0.5 ml) containing 0.3% (vol/vol) Triton X-100 was then added to the filter to release the isotopes into the medium. After 5 h, the detergent extracts were removed. One-milliliter aliquots of buffer were used to rinse the filter. Ten-microliter aliquots were separated for DNA measurements, and radioactivity was determined in the remaining solution and expressed per milligram of DNA.
Cellular levels of DNA. Cellular levels of DNA were measured as described previously (18).
RNA isolation. RNA isolation and preparation of poly(A)+ RNA were described previously (11).
Reverse transcriptase-polymerase chain reaction. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed as described previously (11). We used 1.5 µg of total RNA. The following oligonucleotide primers were used: human P2Y2 receptor (25): forward (sense) 5'-CTC TAC TTT GTC ACC ACC AGC GCG-3' (nucleotides 750-773), reverse (antisense) 5'-TTC TGC TCC TAC AGC CGA ATG TCC-3' (nucleotides 1364-1387); human P2X4 purinergic receptor (GenBank accession no. AF000234): forward (sense) 5'-CTC TGC TTG CCC AGG TAC TC-3' (nucleotides 705-725), reverse (antisense) 5'-CCA GCT CAC TAG CAA GAC CC-3' (nucleotides 1059-1039). The following PCR conditions were applied: P2Y2 receptor: 94°C for 5 min, followed by 30 cycles of 1-min denaturation step at 94°C, 1 min of annealing step at 60°C, and 1 min of extension step at 72°C, followed by 7 min at 720C (expected cDNA length 632 bp); P2X4 receptor: 94°C for 45 s, 35 cycles of 1 min at 94°C, 45 s at 60°C, and 1 min at 72°C, followed by 10 min at 72°C (expected cDNA length 355 bp).
Statistical analysis of data. Data are presented as means ± SD, and significance of differences among means was estimated by Student's t-test. Trends were calculated using GB-STAT V5.3 (Dynamic Microsystems, Silver Spring, MD) and analyzed with ANOVA.
Chemicals and supplies. Anocell (Anocell-10) filters were obtained from Anotec. Fura 2-AM and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM were obtained from Molecular Probes (Eugene, OR). Other chemicals were obtained from Sigma (St. Louis, MO).
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RESULTS |
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ATP phase I and phase II responses.
Micromolar concentrations of ATP stimulate biphasic change in
GTE across CaSki cultures on filters, an acute
transient increase (phase I response) followed by a slower
decrease in permeability (phase II response) (Fig.
1). The biphasic change in permeability is the result of activation of two distinct and independent mechanisms (Fig. 1, inset). Phase I response is the
result of calcium mobilization-dependent cell volume decrease and a
decrease in RLIS; phase II response is mediated by augmented calcium influx via voltage-dependent, dihydropyridine-sensitive calcium channels followed by release of
diacylglycerol and activation of protein kinase C-dependent increase in
RTJ (12, 14). The measured biphasic
change in permeability is therefore the summation of the ATP-induced
decrease in RLIS and increase in
RTJ (Fig. 1, inset; Refs.
12, 15). The hypothesis tested was that
phase I response is triggered by activation of
P2Y2 receptor and phase II response is triggered by activation of P2X4 receptor.
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Uncoupling phase I and phase II responses.
To better understand what triggers phase II responses, we
uncoupled the changes in GTE by uncoupling
calcium signaling. Calcium mobilization and phase I response
were probed with BAPTA (12), whereas calcium influx and
phase II response were probed by lowering extracellular
calcium (15). Effects of BAPTA and low extracellular calcium on changes in cytosolic calcium were determined by two end
points, changes in calcium fluorescence in fura 2-loaded cells and
determinations of 45Ca2+ influx. Fura 2 experiments were done mostly on dispersed cells because ATP changes in
cytosolic calcium have similar time course and magnitude in attached
(Fig. 1) and dispersed (Fig.
2A) cells.
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Effects of purinergic receptor agonists.
The next experiment studied agonist specificity of the
GTE responses during phase I and
phase II responses. Phase I response was
determined in cells bathed in 0.2 mM calcium, whereas phase II response was determined in BAPTA-loaded cells. The order of efficacy for phase I response was ATP > UTP > adenosine 5'-O-(2-thiotriphosphate) (ATP-S) = N6-([6-aminohexyl]carbamoylmethyl)adenosine
5'-triphosphate (A8889) > GTP and for phase II
response was UTP > ATP > GTP = A8889 > ATP-
S
(Table 2). The following agonists had no
significant effect on GTE: adenine, the adenine
derivatives ADP, AMP, and adenosine, the triphosphate nucleosides CTP,
ITP, TTP, and XTP, and the nonhydrolyzable ATP analogs 8-azidoadenosine
5'-triphosphate (A2392), adenosine 5'-O-(2-thiodiphosphate)
(ADP-
S),
,
-methyleneadenosine 5'-triphosphate (AMP-PCP),
,
-imidoadenosine 5'-triphosphate (AMP-PNP),
,
-methyleneadenosine 5'-triphosphate (AMP-CPP),
2-methylthioadenosine 5'-triphosphate (2-MeSATP), and
2',3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (Bz-ATP, in the first 10 min after its addition) (not shown). These
results are similar to data previously reported in cells not probed
with BAPTA or with low extracellular calcium (14).
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Effects of purinergic receptor antagonists.
To further explore the two-receptor hypothesis, cells were treated with
the P2 receptor inhibitors suramin (22) and
pyridoxal phosphate-6-azophenyl-2',4-disulfonic acid (PPADS)
(6) with the experimental design shown in Fig. 2.
Both agents modulated ATP changes in GTE, but
the effects differed for phase I and phase II
responses. Suramin blocked phase I response in a
dose-related manner (Fig. 3A;
IC50 16 ± 2 µM) and attenuated phase II
response (Fig. 3B, IC50 25 ± 3 µM; see
also Fig. 4B). PPADS had
minimal effect on phase I response (Fig. 3A), but
it blocked phase II response in a dose-related manner (Fig.
3B; IC50 10 ± 1 µM).
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Expression of P2X4 and P2Y2 receptor mRNA.
With oligonucleotide primers complementary to human P2X4
receptor, a single cDNA fragment (355 bp) was amplified by RT-PCR from
human CaSki cells (Fig. 7). Sequence
analysis of the cloned segment revealed homologies of 99% (sense and
antisense) with the human P2X4 (the differences were
sequence errors; not shown). Figure 7 also shows, for comparison, the
expression of a 632-bp cDNA fragment corresponding to the human
P2Y2 receptor in lysates of CaSki cells, which confirms our
previous study (11).
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DISCUSSION |
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ATP-induced changes in paracellular permeability across cultured
cervical epithelia can be described in terms of activation of two
distinct types of P2 receptor mechanisms (present results and Refs.
12, 14, 15, 17).
Phase I response is activated by a decrease in
RLIS; it is mediated by calcium
mobilization-dependent cell volume decrease and can be mimicked by
ionomycin. Phase I response can be characterized as
interaction of ATP with a single class effector with ATP
EC50 of 25 µM and an agonist profile of ATP > UTP > ATP-S = A8889 > GTP. Suramin blocked
phase I response and ATP-induced calcium mobilization
without significantly affecting phase II response and
calcium influx. In human cervical cells calcium mobilization is
necessary and sufficient to stimulate phase I-like increase
in permeability (15). In other cell types, calcium
mobilization is usually induced by activation of P2Y receptors (26) and suramin is a previously described P2Y receptor
antagonist (3, 6, 22). Because human cervical epithelial
cells express P2Y2 receptor mRNA (present results and Ref.
11), it is likely that phase I response, but
not phase II response, is mediated by activation of a
P2Y2 receptor.
In contrast to phase I response, phase II
response is mediated by calcium influx-dependent diacylglycerol
activation of protein kinase C, and it can be mimicked by
1,2-dioctanoyl-sn-diglycerol (diC8), which activates protein
kinase C-dependent increase in RTJ (present results and
Refs. 14, 15). Increases in cytosolic calcium
per se, such as those induced by ionomycin or histamine (15), cannot induce phase II decrease in
GTE, indicating that ATP-induced calcium influx
is necessary to stimulate phase II response. Phase
II response could be described as interaction of ATP with a single
class effector that differs in potency and agonist profile from
phase I response, with ATP EC50 of 2 µM and agonist profile of UTP > ATP > GTP = A8889 > ATP-S. Direct determinations of calcium influx by measurements of
45Ca2+ entry agreed with the results of the
fura 2 experiments and support the conclusion that phase II
response is mediated by calcium influx. The kinetic profiles of
ATP-induced 45Ca2+ influx and ATP phase
II decrease in GTE indicated interactions with a single class of effectors,
of 2 min, and saturable
concentration-dependent effects (ATP EC50 of 2 µM).
Phase II response and ATP-augmented calcium influx could be blocked with PPADS. PPADS had only mild effect on phase I response and ATP-induced calcium mobilization. Because PPADS is a more selective P2X antagonist than suramin (26), the present results suggest that phase II response is mediated by a P2X receptor. In the calcium influx experiments PPADS attenuated, but did not entirely block, calcium entry after treatment with ATP. A possible explanation is that in addition to activation of PPADS-sensitive calcium channels, ATP also stimulates store-operated capacitative calcium influx via PPADS-insensitive calcium channels to prevent depletion of calcium from intracellular stores (15).
It was previously suggested that P2 purinoceptor antagonists exert their effects by inhibition of the enzymatic breakdown of extracellular ATP and ADP (32). The present results in human cervical cells refute this explanation because ADP, AMP, and adenine had no effect on permeability.
Phase II response depended on ATP-induced calcium influx (present results and Refs. 14, 15). Because P2X receptors are ATP-gated calcium channels and activation by ATP of P2X receptors stimulates calcium influx (26), our working hypothesis was that phase II response is triggered by activation of P2X receptor. The following experimental findings suggest that in human cervical cells phase II response is mediated by the P2X4 receptor. First, human cervical epithelial cells express P2X4 mRNA. Second, only UTP and GTP elicited an appreciable phase II response, whereas AMP-CPP (agonist of P2X1 and P2X3 receptors), AMP-PCP (agonist of P2X1 receptor), and 2-MeSATP (agonist of P2X1, P2X2, P2X3, and P2X5 receptors) (24) had no effect on permeability (present results and Ref. 14). In addition, the selective P2X7 receptor agonist Bz-ATP (26) did not stimulate changes in cytosolic calcium or in GTE in the first 5-10 min after its addition (not shown), indicating that phase I and phase II responses are not mediated by P2X7 receptor. Third, the P2X receptor antagonist PPADS, but not the P2Y receptor antagonist suramin, blocked phase II response and ATP-induced calcium influx. The IC50 of 10-25 µM is significantly higher than levels reported for blocking rat P2X1, P2X2, P2X3, and P2X5 receptors and lower than levels for blocking rat P2X4 receptor (24) but is similar to levels recently reported for blocking the human P2X4 receptor (7).
On the basis of these results we propose that in human cervical epithelial cells phase I response is mediated by the P2Y2 receptor and phase II response by the P2X4 receptor. Both receptors are localized predominantly, but not exclusively, on the apical membrane (14). Activation of P2Y2 stimulates acute calcium mobilization that triggers cell volume decrease and decrease in RLIS. Activation of the P2X4 receptor stimulates calcium influx via a complex mechanism that involves voltage-dependent, dihydropyridine-sensitive calcium channels, followed by release of diacylglycerol and activation of protein kinase C-dependent increase in RTJ (12, 14). The latter mechanism is not well understood, but it may involve protein kinase C-dependent phosphorylation of tight junctional proteins. The present data may have clinical significance, given the potential role of ATP regulation of cervical paracellular permeability for human fertility, contraception, and health (10, 13, 15). Improving our understanding of purinergic regulation of permeability may provide drugs that can target specific signaling pathways and effectors of paracellular resistance in the uterine cervix.
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ACKNOWLEDGEMENTS |
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The technical support of Kim Frieden, Brian De Santis, and Dipika Pal is acknowledged.
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FOOTNOTES |
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The study was supported by National Institutes of Health Grants HD-00977, HD-29924, and AG-15955.
Address for reprint requests and other correspondence: G. I. Gorodeski, Univ. MacDonald Women's Hospital, Univ. Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106 (E-mail: gig{at}po.cwru.edu).
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. Section 1734 solely to indicate this fact.
Received 2 July 2001; accepted in final form 5 September 2001.
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