Regulation of transcervical permeability by two distinct P2 purinergic receptor mechanisms

George I. Gorodeski

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma S) N6-([6-aminohexyl]carbamoylmethyl)adenosine 5'-triphosphate (A8889) > GTP and UTP > ATP > GTP = A8889 > ATP-gamma 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Omega  · 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Delta 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 (Delta PD; lumen negative) as GTE = Delta I/Delta 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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Effects of extracellular ATP on transepithelial electrical conductance (GTE, ) and cytosolic calcium (Cai, open circle ) across CaSki cells on filters. Determinations of GTE were done in Ussing chambers, and measurements of Cai were done on fura 2-loaded cells attached on filters in custom-made fluorescence chamber (see METHODS). Shown are actual tracings: filter with cells was mounted in Ussing chambers; after stabilization, ATP (50 µM) was added from a 1,000× stock (pH 7.2) to luminal and subluminal solutions. After GTE stabilized (~10 min), the filter was transferred to the fluorescence chamber; cells were continuously irrigated for 60 min in fresh medium lacking ATP to resensitize the purinergic receptors and then loaded with fura 2. After an additional 60 min, Cai measurements were done. The acute transient increase in GTE is designated phase I response; the late persistent decrease in GTE is designated phase II response. Experiments were repeated 6 times (3 times with Cai measurements first, followed by GTE determinations). Inset, model describing the ATP-induced changes in GTE across CaSki cultures in terms of 2 independent effects (phases I and II) that differ in time course, direction, and magnitude.

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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Fig. 2.   Modulation of ATP changes in Cai (A) and GTE (B) by chelating Cai with BAPTA or by lowering extracellular calcium (Cao). A: dispersed fura 2-loaded CaSki cells were treated with ATP (50 µM, arrows), and changes in Cai were determined as described in METHODS. Experiments were done on control cells (C) maintained in normal calcium (1.2 mM) or on cells that were shifted to 0.2 mM calcium by adding aliquots from concentrated EGTA solution (300×, pH 7.2) 5 min before experiments. In some experiments cells loaded with fura 2 were also preincubated with 10 µM BAPTA-AM for 15 min and then treated with ATP. B: experiments were done as in A on CaSki cells attached on filters. Experiments in each panel were repeated 3-5 times, and results are summarized in Table 3.

In cells loaded with the calcium chelator BAPTA, ATP evoked only slow calcium influx (Fig. 2A; Table 1) and a slow decrease in GTE (i.e., phase II response; Fig. 2B, Table 1). Lowering extracellular calcium increased baseline GTE [by abrogation of RTJ (19)] and blocked phase II response (Fig. 2B; Table 1) without significantly affecting ATP-induced calcium mobilization or the peak of phase I response (Fig. 2; Table 1). In cells loaded with BAPTA and maintained in low extracellular calcium, ATP caused no significant change in cytosolic calcium or GTE (Fig. 2; Table 1). These results show that BAPTA and low extracellular calcium can uncouple calcium signaling and phase I and phase II responses.

                              
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Table 1.   Modulation by low extracellular Ca and BAPTA of ATP effects on cytosolic Ca and GTE

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-gamma S) = N6-([6-aminohexyl]carbamoylmethyl)adenosine 5'-triphosphate (A8889) > GTP and for phase II response was UTP > ATP > GTP = A8889 > ATP-gamma 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-beta S), beta ,gamma -methyleneadenosine 5'-triphosphate (AMP-PCP), alpha ,gamma -imidoadenosine 5'-triphosphate (AMP-PNP), alpha ,beta -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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Table 2.   Agonist induced changes in GTE

The general pattern of agonist efficacy of ATP approx  UTP >> 2-MeSATP, AMP-CPP, AMP-PCP suggests that both phase I and phase II responses are mediated by P2 receptors (26). The effects do not require hydrolysis of ATP because the poorly hydrolyzable analog ATP-gamma S elicited both responses. The individual rank of agonist efficacy for phase I and phase II responses showed significant differences and is consistent with the expression of two different P2 receptors in cervical cells, one that produces phase I response and one that produces phase II response.

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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Fig. 3.   Effects of pyridoxal phosphate-6-azophenyl-2',4-disulfonic acid (PPADS) and suramin on phase I (A) and phase II (B) responses. Filters containing cells were mounted in Ussing chambers, and drugs were added from concentrated (500×) stocks for the indicated final concentrations 5 min before ATP was added. Effects on phase I response were determined in cells bathed in 0.2 mM calcium and are reported as %peak increase in GTE compared with control cells. Effects on phase II response were determined in BAPTA-loaded cells and are reported relative to GTE nadir in control cells (see Fig. 2). Shown are means ± SD of 3-7 filters at each point. Data of the means in A and B for each of the 3 drugs were fitted into a modified Hill equation: G = Gmax · 1/[1 + (IC50/[Ag])nH] + Gmin · {1 - 1/[1 + (IC50/[Ag])nH]}, where G is the measured GTE, Gmax and Gmin are the maximal (100%) and minimal GTE, respectively, IC50 is the drug concentration that produces half-maximal effect, [Ag] is the concentration of the drug, and nH is the Hill coefficient. Trends of all 4 curves were significant at P < 0.01. Values of nH for the means were 1.9 and 1.4 in A and 1.2 and 1.8 in B for PPADS and suramin, respectively.



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Fig. 4.   Modulation of ATP changes in Cai (A) and GTE (B) by chelating Cai with BAPTA and by treatments with PPADS or suramin. Experiments were done as in Fig. 2 on cells bathed in normal calcium (1.2 mM). Suramin or PPADS was added as indicated from concentrated (500×) stocks for final concentrations of 75 µM 5 min before ATP was added. Arrows, addition of ATP (50 µM) to luminal and subluminal solutions. Experiments were repeated 3 times.

Suramin and PPADS also modulated ATP effects on cytosolic calcium. In fura 2-loaded cells suramin, at a submaximal concentration of 75 µM (Fig. 3A), abrogated ATP-induced calcium transients (i.e., calcium mobilization) but had little effect on the slow, sustained increase in cytosolic calcium (i.e., calcium influx). Coloading cells with BAPTA abolished ATP-induced calcium mobilization (Figs. 4A and 5A). In fura 2-loaded cells PPADS, at a submaximal concentration of 75 µM (Fig. 3B), did not significantly affect calcium mobilization, but it abrogated calcium influx; in cells coloaded with BAPTA and treated with PPADS, ATP did not elicit significant changes in cytosolic calcium or in GTE (Figs. 4A and 5A).


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Fig. 5.   Modulation of ATP changes in Cai (A) and GTE (B) by PPADS or suramin. A: dispersed fura 2-loaded CaSki cells were treated with ATP at the indicated concentrations. Changes in calcium transients (T) were determined in cells shifted to 0.2 mM calcium by adding aliquots from a concentrated EGTA solution (300×, pH 7.2). Changes in the late sustained increase in Cai (SI) were determined in cells preincubated with 10 µM BAPTA-AM for 15 min. Net changes in Cai are expressed relative to baseline Cai (95 ± 7 nM in cells incubated in 1.2 mM and 70 ± 11 in cells incubated in 0.2 mM calcium). B: cultured CaSki cells attached on filters were treated with ATP at the indicated concentrations. Changes in phase I response (top) were determined in cells shifted to 0.2 mM calcium by adding aliquots from concentrated EGTA solution (300×, pH 7.2). Changes in phase II response were determined in cells preincubated with 10 µM BAPTA-AM for 15 min. Effects on phase I and II responses are reported as %peak increase or decrease (nadir), respectively, in GTE relative to changes in GTE in control cells (see Fig. 2). PPADS or suramin was added as indicated from concentrated (500×) stocks for final concentrations of 75 µM 5 min before ATP was added. Control cells were treated with vehicle. Experiments were repeated 3 times; variability in A and B ranged from 5% to 15%. Data of the means in A and B for each curve were fitted into a modified Hill equation (see Fig. 3). Trends of all 12 curves were significant at P < 0.01. Values of nH in A were 1.1, 1.4, and 1.6 for T-C, T-PPADS, and T-suramin and 1.2, 1.7, and 1.8 for SI-C, SI-suramin, and SI-PPADS, respectively. Values of nH in B were 1.2, 1.3, and 1.6 for phase I C, T-PPADS, and suramin and 1.1, 1.6, and 1.8 for phase II C, suramin, and PPADS, respectively.

Figure 5 summarizes the effects of ATP, PPADS, and suramin on cytosolic calcium and GTE (Fig. 5B). ATP effects on cytosolic calcium (Fig. 5A) and phase I and phase II responses (Fig. 5B) were concentration dependent and saturable. Calculated mean EC50 was 3 µM for calcium mobilization and 4 µM for the sustained calcium increase; corresponding values for phase I and phase II responses were 25 and 2 µM, respectively (Fig. 5B). These results confirm previous studies done with a different experimental design (14). Suramin blocked ATP-induced calcium mobilization (Fig. 5A) and phase I response (Fig. 5B), and it attenuated ATP-sustained increase in cytosolic calcium by ~15%, without significantly changing ATP EC50. Suramin also attenuated phase II response by ~10%, and it increased ATP EC50 from 2 to 9 µM (Fig. 5B; P < 0.05). PPADS blocked ATP-sustained increase in cytosolic calcium and phase II response and attenuated calcium mobilization and phase I response by 25% and 10%, respectively, without significantly changing ATP EC50 (Fig. 5). The effects of ATP, ATP + PPADS, and ATP + suramin could be described in terms of the Hill equation with Hill coefficient values of 1.1 ± 0.1 for ATP, 1.4 ± 0.1 for ATP + PPADS, and 1.6 ± 0.2 for ATP + suramin effects. These results indicate that ATP interacts with one class of effectors, but PPADS and suramin block ATP effects by a more complex type of interaction.

The effects of PPADS and suramin in fura 2-loaded cells on ATP-induced calcium influx were confirmed by 45Ca2+ influx experiments in attached cells. In control cells, ATP stimulated 45Ca2+ influx in a time-related manner, with net accumulation of ~8 pmol/mg DNA after 6 min (Fig. 6A). The results of four experiments yielded a mean (±SD) best fit value for the time constant tau  of 2.1 ± 0.6 min (P < 0.01; Fig. 6A). This value is similar to that of phase II response (14, 15). Pretreatment with suramin had a mild effect on 45Ca2+ accumulation, but PPADS attenuated it by ~75% and increased tau  to 7.3 ± 1.6 min (P < 0.01; Fig. 6A).


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Fig. 6.   Time (A)- and dose (B)-response effects of ATP, PPADS, and suramin on 45Ca2+ influx; the assay is described in METHODS. Experiments were done on control cells or cells treated with 75 µM PPADS or suramin 5 min before ATP was added. Net accumulation of 45Ca2+ was determined by subtracting cellular 45Ca2+ in parallel cultures treated with the vehicle from that in cultures treated with ATP and expressed per mg of cellular DNA. Values are means ± SD of 4 filters at each point. In A, the time-related increase in net accumulated 45Ca2+ could be fitted by the equation [45Ca]t = [45Ca]max + ([45Ca]min - [45Ca]max) · e-t/tau , where [45Ca]t is the 45Ca2+ concentration ([45Ca]) at time t, [45Ca]max is maximal [45Ca], [45Ca]min is baseline (minimal) [45Ca] at t = 0 (i.e., before ATP was added), and tau  is the time constant. All 3 trends were significant at P < 0.01. In B, net accumulated 45Ca2+ was determined 6 min after ATP was added. Data of the means in B for the 3 curves were fitted into a modified Hill equation (see Fig. 3). Trends of all 3 curves were significant at P < 0.01. Values of nH were 1.1, 1.4, and 1.1 for ATP, ATP + suramin, and ATP + PPADS, respectively.

The effect of ATP on 45Ca2+ influx was concentration dependent; augmented 45Ca2+ influx was observed already at 0.4 µM ATP, reaching saturation at ~8 µM, with calculated EC50 for ATP of 3 ± 1 µM (Fig. 6B). PPADS and suramin attenuated the ATP effect by 80% and 15%, respectively, and both increased ATP EC50 to ~10 µM (Fig. 6B). The effect of ATP on 45Ca2+ influx could be described in terms of the Hill equation with Hill coefficient values of 1.0 ± 0.1 for ATP, 1.3 ± 0.2 for ATP + PPADS, and 1.7 ± 0.3 for ATP + suramin. These results indicate that ATP interacts with one class of effectors in stimulating 45Ca2+ influx, but PPADS and suramin abrogate ATP effect by a more complex type of interaction.

Collectively, the results shown in Figs. 3-6 indicate that suramin blocks mainly calcium mobilization and phase I response, whereas PPADS blocks mainly calcium influx and phase II response. In view of previously reported effects of suramin and PPADS on purinergic receptors (3, 6, 22), these results suggest involvement of P2Y2 and P2X purinergic receptors, respectively, in the phase I and phase II responses to ATP.

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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Fig. 7.   CaSki cells express mRNA of P2X4 and P2Y2 receptors: RT-PCR results using total RNA extracted from lysates of CaSki cells. Oligonucleotide primers complementary to cloned human P2X4 and P2Y2 receptors were used to amplify single cDNA fragments of 355 and 632 bp, respectively. Experiments were repeated 5 times. M, markers.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma 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-gamma 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, tau  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.


    ACKNOWLEDGEMENTS

The technical support of Kim Frieden, Brian De Santis, and Dipika Pal is acknowledged.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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