Clminus currents activated via purinergic receptors in Xenopus follicles

Rogelio O. Arellano1, Edith Garay1, and Ricardo Miledi1,2

1 Centro de Neurobiología, Universidad Nacional Autónoma de México, Queretaro, Queretaro 76001, Mexico; and 2 Laboratory of Cellular and Molecular Neurobiology, University of California, Irvine, California 92697-4550

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Ionic currents elicited via purinergic receptors located in the membrane of Xenopus follicles were studied using electrophysiological techniques. Follicles responded to ATP-activating inward currents with a fast time course (Fin). In Ringer solution, reversal potential (Erev) of Fin was -22 mV, which did not change with external substitutions of Na+ or K+, whereas solutions containing 50 or 5% of normal Cl- concentration shifted Erev to about +4 and +60 mV, respectively, and decreased Fin amplitude, indicating that Fin was carried by Cl-. Fin had an onset delay of ~400 ms, measured by application of a brief jet of ATP from a micropipette positioned near the follicle (50 µm). Fin was inhibited by 50% in follicles pretreated with pertussis toxin. This suggests a G protein-mediated receptor channel pathway. Fin was mimicked by 2-MeSATP and UTP, the potency order (half-maximal effective concentration) was 2-MeSATP (194 nM) > UTP (454 nM) > ATP (1,086 nM). All agonists generated Cl- currents and displayed cross-inhibition on the others. Fin activation by acetylcholine also cross-inhibited Fin-ATP responses, suggesting that all act on a common channel-activation pathway.

chloride channels; Xenopus oocytes; UTP receptors; follicular cells

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

xenopus laevis FOLLICLES possess cholinergic (1, 17, 18) and purinergic (3, 19, 20) receptors in their membrane. The stimulation of these receptors evokes complex ionic current responses that involve activation of various channel types. Electrical responses evoked by acetylcholine (ACh) have their origin in the oocyte membrane and/or in the follicular cells, which maintain a strong metabolic and electrical coupling with the oocyte via gap-junction channels (7). Because of these junctions, currents arising in either compartment can be monitored by electrodes inserted into the oocyte (1, 18, 33). Purinergic receptors and the current response they generate are localized preferentially in the follicular cells (3). The follicular cell responses elicited by ACh or purinergic agents usually show two inward current components that flow through different types of ionic channels (1, 3). From their time course, these currents were named Sin (slow and smooth) and Fin (fast). Their intrinsic characteristics and localization in follicular cells differentiate them clearly from depolarizing currents arising in the oocyte itself (for a detail description, see Ref. 3).

Characterization of follicular cell membrane currents and receptors is essential for a comprehensive understanding of ovarian physiology. In particular, this information is necessary to elucidate the physiological role played by the membrane molecules involved and the intracellular mechanisms activated. The study of the mechanisms of activation of Fin by purinergic and muscarinic receptors may also offer information relevant to the generation of osmodependent Sin, which are probably involved in the regulation of cell volume (1, 2).

Although some of the Fin characteristics have been reported previously (e.g., 3, 16, 19, 20), their detailed and unambiguous analysis had been somewhat hampered because of the requirement to maintain coupling between follicular cells and oocyte in vitro. This is obviously necessary to effectively preserve responses that originate in the follicular cells. Continuing with the study of the follicle properties, we have found better conditions for their maintenance and electrical recording (1-3). Here we present a more detailed characterization of Fin elicited by purinergic agents and of the receptors and membrane mechanisms activated.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Xenopus laevis frogs were obtained from Xenopus I (Ann Arbor, MI). The ovary lobules were surgically removed in sterile conditions from frogs anesthetized by hypothermia and killed by decapitation and pithing. The lobules were placed in sterile unsupplemented modified Barth's medium containing (in mM) 88 NaCl, 0.2 KCl, 2.4 NaHCO3, 0.33 Ca(NO3)2, 0.41 CaCl2, 0.82 MgSO4, 0.88 KH2PO4, and 2.7 Na2HPO4 (pH 7.4), with 70 µg/ml gentamicin.

Follicles (stage VI, Ref. 11) were dissected as epithelium removed, where the follicle's inner epithelia, together with thecal blood vessels (and other adjoining cell types), were separated using sharp forceps. This procedure leaves the follicular cell basement membrane, thus providing protection and a natural environment to the follicular cells. Images with scanning electron microscopy have shown that the basement membrane in follicles dissected in this way appears as a layer of loosely woven collagenous bundles and tangles. Immediately beneath is the layer of follicular cells (see plates 1, A and B, in Ref. 25). This dissection facilitates electrode insertion, improves the stability of electrophysiological recording, and simplifies the interpretation of results by eliminating the possible participation of epithelium and other surrounding thecal tissues in the responses. Moreover, it also seems to improve the recording of native responses to several agonists, apparently by eliminating a diffusional barrier introduced by the external layers. Epithelium-removed follicles were incubated (18-20°C) in sterile modified Barth's medium supplemented with glucose (5 mM) and fetal bovine serum (0.1-0.2%). Under these conditions, follicular cell-oocyte electrical coupling and follicular responses can be maintained for >15 days. In some experiments, complete oocyte defolliculation was performed by collagenase treatment [0.7 mg/ml, 45 min in normal Ringer (NR) solution] and subsequent manual removal of any remaining follicular layers using sharp forceps, as already described (3, 25).

Follicular electrical responses were monitored using a two-electrode voltage clamp (21). Osmodependent follicular Sin (1-3) were minimized by superfusing the follicles with NR solution containing (in mM) 115 NaCl, 2 KCl, 1.8 CaCl2, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.0). Unless otherwise stated, follicles were voltage clamped at -60 mV, and drugs were applied by superfusion. Ionic substitutions in NR solution were made as follows: 1) all NaCl by tetraethylammoniun chloride (TEA-Ringer), 2) NaCl by NaI, NaBr, or NaSCN, and 3) 50% NaCl by KCl. Ringer solutions with reduced Cl- concentration (50 or 5%) were prepared by substituting NaCl with Na2SO4 and compensating osmolarity with sucrose. In some experiments, ATP (1-100 µM in NR) was applied extracellularly by electronically controlled pressure pulses from a micropipette positioned close (~50 µm) to the follicle. By use of this method, the superfusion rate was lowered (from 10 to 2 ml/min) to favor receptor activation, since otherwise the agonist is washed away before any current can be detected. The same injection apparatus was used for intracellular delivery of ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) as described elsewhere (1, 22).

Follicle-stimulating hormone (FSH) and forskolin were purchased from Calbiochem (La Jolla, CA). Suramin, pertussis toxin, 2-methylthio-ATP trisodium salt (2-MeSATP), and beta gamma -methylene ATP (beta gamma -MeATP) were from RBI (Natick, MA). TEA chloride was obtained from Baker (Phillipsburg, NJ). All other compounds [collagenase type I, ATP, UTP, ADP, AMP, ACh, angiotensin II (ANG II), EGTA, and BAPTA] were from Sigma Chemical (St. Louis, MO).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Ionic basis of follicle ATP-elicited fast responses. In the present study, 19 of 20 frogs had follicles that responded to extracellular applications of ATP (0.1-100 µM) eliciting Fin. As already reported for other follicular responses, large variations in current amplitude between donors were observed (e.g., 1, 3, 33). However, the Fin amplitudes from follicles of a given frog were more consistent. Thus follicles (n = 183) from 19 frogs that were first tested at the beginning of an experiment with 50 µM ATP and during days 2 and 3 of incubation can be divided in two groups, follicles that were low responsive (Fin in 20- to 300-nA range) and highly responsive (301- to 2,000-nA range) to ATP. The first group included 23% of the follicles and showed Fin of 159 ± 96 nA (mean ± SE), and the second group, which accounts for the remaining 77% of the follicles, had a mean amplitude of 819 ± 397 nA. The latter, the highly responsive follicles, were used preferentially for this study. Nevertheless, most of the results were confirmed in low-responsive follicles. Therefore, there was no doubt that the differences were simply due to different response amplitudes and not to qualitative differences in the types of channels activated.

We also confirmed the already described general characteristics of Fin (cf. Refs. 1, 3). For example, 1) all follicular Fin elicited by ATP (50 µM) and other purinergic agonists (see below) were eliminated by defolliculation; 2) they were not affected by oocyte loading with EGTA or BAPTA (100-200 pmol/oocyte), which nevertheless eliminated the Ca2+-dependent Cl- currents that arise in the oocyte itself; 3) their onset delay did not vary greatly with agonist concentration; and 4) they were not osmodependent currents. In this study, 23 defolliculated oocytes from 9 frogs did not show any response to ATP, even at concentrations of 0.5-1 mM. Although some batches of frogs had defolliculated oocytes that responded to ATP with a small inward current of 1-3 nA, this has not yet been characterized (Miledi and Arellano, unpublished results). The reversal potential (Erev) of purinergic Fin in both high- and low-responsive follicles was measured using two methods. In method A, ATP (5-10 µM) was applied to the follicle at 15-min intervals while it was maintained at different holding potentials (Fig. 1A). As a variation of this method, a micropipette containing ATP (10-100 µM in NR) was positioned close to the follicle's animal pole, and ATP was pressure ejected, activating reproducible Fin at intervals of at least 60 s (e.g., see Fig. 3B). Fin were again measured at various holding membrane potentials. Method B consisted of changing the membrane potential from +40 to -100 mV in 20-mV steps with durations of 80 ms before (control membrane current) and during the peak of the Fin activated by superfusion of ATP (50 µM). For every voltage step, the control membrane currents were subtracted from those obtained during ATP-elicited currents, and these values were plotted as in Fig. 1B. The Erev obtained with each procedure was as follows: method A, -23 ± 5 mV, 25 follicles from 6 frogs; and method B, -21.5 ± 4.5 mV, 34 follicles from 5 frogs. Together, Erev gave a mean value of -22 mV, close to the equilibrium potential for Cl- in Xenopus follicles (about -20 mV; Ref. 18). This already indicated that the Fin activated by ATP were carried mainly by Cl-, and, to confirm this, the Erev was estimated (using method B) in solutions containing 50 or 5% of the normal Cl- concentration (Fig. 1B). When 50% of Cl- was substituted with SO<SUP>2−</SUP><SUB>4</SUB>, the Erev was +4.9 ± 3 mV (8 follicles, 3 frogs), whereas 95% of Cl- substitution (all NaCl by Na2SO4) shifted the Erev to +60 ± 6.8 mV (10 follicles, 3 frogs). Both low-Cl- solutions produced a clear reduction in Fin amplitude in the whole voltage range tested (Fig. 1B). Erev obtained in those solutions were as expected for a membrane selectively permeable to Cl-, as confirmed by calculating the theoretical equilibrium potential, assuming a value of 60 mM for intracellular Cl- concentration (18) and substituting Cl- concentration values in the Nernst equation (Fig. 1B, inset). Contrary to this, substitution of Na+ with TEA-Ringer (Fig. 1C) or 50% Na+ with K+ (not shown) did not alter the Erev (16 follicles, 4 frogs).


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Fig. 1.   Ionic basis of fast inward currents (Fin) elicited by ATP. A: Fin elicited by ATP in a follicle held at 0, -20, -40, and -60 mV. Interval between ATP applications was 15 min. In this and subsequent records, agonists were applied during times indicated by bars at top, and +20-mV depolarizing pulses served to monitor follicular membrane conductance. B: relationship between membrane potential (Vm) and ATP-Fin in normal Ringer solution (NR; bullet ) and in solutions containing 50% (triangle ) or 5% (open circle ) Cl-. Reversal potentials (Erev) in later solution were estimated from linear regressions of data between -20 to +40 mV. Inset: relationship between Erev and external Cl- concentration; line is Nernst relationship assuming a cytoplasmic Cl- concentration of 60 mM. Data represent means ± SE of 4-7 follicles in each condition. C: current-voltage (I-V) relationship for ATP-Fin in NR (bullet ), tetraethylammonium chloride (TEA)-Ringer (square ), and solutions in which NaCl was substituted with NaSCN (star ), NaI (open circle ), or NaBr (triangle ) (3-5 follicles each condition).

The permeability to other anions was also investigated. Fin activated by ATP showed Erev values of -51 ± 6, -45 ± 5, and -29 ± 3 mV in solutions containing SCN-, I-, or Br-, respectively, as principal anions (Fig. 1C, 6-8 follicles for each anion substitution, 2-3 frogs). Moreover, in these solutions, the Fin increased between 70 and 150%, all of which suggests that Fin channels were more permeable to those ions than to Cl-.

Fin responses elicited by purinergic agonists. From a group of purinergic agonists (ADP, AMP, and beta gamma -MeATP, not shown), it was found that ATP-elicited Fin were strongly mimicked by UTP or 2-MeSATP, which is in accordance with previous observations (3, 16). All three agonists behaved as full agonists eliciting large Fin. Dose-response relationships for each agonist were determined in follicles held at -60 mV, and the curves gave half-maximal effective concentration values of 1,086 ± 200 nM for ATP and 454 ± 80 and 194 ± 50 nM for UTP and 2-MeSATP, respectively (5-7 follicles, 3 frogs; Fig. 2A).


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Fig. 2.   Fin elicited by purinergic agonists. A: dose-response relationship for Fin elicited by ATP (bullet ), UTP (triangle ), and 2-methylthio-ATP (2-MeSATP; black-lozenge ) in follicles held at -60 mV. Follicles were different for every agonist, and data are averages for 3-4 follicles. Curves show fit to equation: Fin/Fin,max = [A1 - A2/1 + ([agonist]/EC50)n] + A2. Half-maximal effective concentrations (EC50) and n (in parentheses) were 1,086 ± 200 nM (0.7) for ATP, 454 ± 80 nM (1) for UTP, and 194 ± 50 nM (0.85) for 2-MeSATP. B: Fin elicited by ATP, 2-MeSATP, or UTP (10 µM), all in 1 follicle held at -15, -25, or -60 mV (15-min application intervals). Similar results were obtained in 8 follicles from 3 frogs. C: I-V relationship for Fin elicited by UTP and 2-MeSATP in NR (black-triangle and bullet , respectively) or in 5% Cl- (triangle  and open circle , respectively).

The Fin elicited by the three agonists were uniform in amplitude among follicles from the same frog. For example, in follicles (n = 7) from one donor, the amplitude of the Fin activated by ATP (10 µM) was 783 ± 151 nA and those activated by UTP and 2-MeSATP (10 µM) were 842 ± 174 and 748 ± 135 nA, respectively. In a different frog with low-responsive follicles (n = 6), the Fin elicited by ATP, UTP, and 2-MeSATP (all at 10 µM) had amplitudes of 189 ± 34, 202 ± 50, and 184 ± 45 nA, respectively. In 12 frogs tested with the 3 agonists, in no case were the Fin amplitudes dissimilar when follicles from the same frog were compared. For these same group of donors, a clearly different result was obtained when the currents elicited by ACh and ATP were compared. In this case, follicles could have Fin with very different amplitudes, although in two frogs their follicles (n = 7) had similar Fin amplitudes for both agonists. For instance, in follicles (n = 12) from one frog ACh (50 µM) activated Fin of 118 ± 36 nA, whereas ATP (50 µM) gave 1,234 ± 244 nA; a similar divergence in current amplitudes was obtained in follicles from five other frogs. In an extreme example of this, in one frog, ACh-elicited Fin were practically absent (Fig. 4, B and C), whereas ATP generated robust (1,496 ± 389 nA) Fin (8 follicles). In follicles (n = 13) from three frogs showing heterogeneity between the Fin amplitudes, ACh was more effective than ATP in eliciting the current. Thus these experiments indicated that the responses to ATP and similar agonists are not independent. However, suramin (100 µM), which blocks P2-type purinergic receptors, inhibited the Fin responses elicited by ATP, 2-MeSATP, or UTP (all at 10 µM) by 53 ± 4, 56 ± 3, and 28 ± 6%, respectively (4-7 follicles for each agonist, 2-3 frogs).

Because it has been suggested that ATP and UTP activate different sets of ionic channels in follicles (at least those involved in generating slow inward currents) from the channels opened by 2-MeSATP (16), we analyzed the Erev values for Fin elicited by ATP (5-50 µM), UTP (5-50 µM), or 2-MeSATP (5-20 µM) in the same follicles (Fig. 2, B and C). We also studied the currents evoked by UTP and 2-MeSATP in solutions with low concentration of Cl- and compared them with Fin elicited by ATP (Fig. 2C). Our results suggest strongly that Fin, activated by either UTP or 2-MeSATP, were carried mainly by Cl-, similar to what was found for the currents elicited by ATP (cf., Fig. 1). For instance, 1) the Erev of Fin was the same for all three agonists (-23 ± 4 mV for ATP, -23.6 ± 3.5 mV for UTP, and -22 ± 4.6 mV for 2-MeSATP), independent of the method used for their estimation (Fig. 2, B and C), and all corresponded to the equilibrium potential for Cl- in Xenopus follicles. 2) Erev of Fin elicited by UTP and 2-MeSATP shifted as predicted by the Nernst equation, for a decrease in extracellular Cl- concentration (Fig. 2C). For example, Erev values of Fin elicited by UTP and 2-MeSATP in Ringer solution containing 50% Cl- were +3.5 ± 3 and +4.6 ± 2 mV, respectively (4-6 follicles, 3 frogs), whereas, in solutions with only 5% Cl-, values were +58 ± 6 and +61 ± 4 mV, respectively (5-7 follicles, 3 frogs). In addition, Erev for Fin activated by either UTP (50 µM) or 2-MeSATP (20 µM) (3-4 follicles, 2 frogs) did not show differences in TEA-Ringer.

Therefore, all these results strongly indicate that the purinergic receptors of the follicular cells are potently activated by ATP, UTP or 2-MeSATP and that this activation leads to opening of the same type of ionic channels, which are permeable mainly to Cl-.

Purinergic receptor-Fin channel-coupling pathway interactions. Further evidence suggesting that all the three agonists activate the same set of Cl- channels derives from experiments showing cross-inhibition between the currents elicited by purinergic agonists (Fig. 3A). In these experiments, a nearly saturating concentration of ATP (20-50 µM) was applied ~1 min before application of one of the other agonists (20-50 µM), which then produced responses of only ~5% of their control (11 follicles, 4 frogs). Similarly, UTP or 2-MeSATP cross-inhibited the responses elicited by a subsequent application of ATP (same follicles), and, importantly, cross-inhibition was also observed for the Fin elicited by ACh (20-100 µM; see below). Together these results strongly suggest that ATP, UTP, and 2-MeSATP activate the same receptor-Cl- channel-coupling pathway.


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Fig. 3.   Interactions between Fin elicited by purinergic agonists and currents evoked by follicle-stimulating hormone (FSH) and ANG II (AII). A: cross-inhibition of Fin elicited by ATP (50 µM) on those generated by UTP (50 µM) or 2-MeSATP (20 µM), and Fin inactivation caused by latter agonists on ATP (same concentrations). All records are from same follicle. Similar results were obtained in 10 additional follicles (4 frogs). B: records are from 2 different follicles exposed to either FSH or ANG II while stimulated repeatedly with short pulses (100 ms) of ATP (5 µM) ejected approximately every 90 s using a micropipette positioned at ~50 µm from follicle (ATP currents are indicated by arrowheads during ANG II oscillatory response). Inset: Fin elicited by ejection of ATP (5-50 µM) in 9 different follicles (4 frogs), superimposed to show their onset delay and time course.

To investigate in more detail the mechanism utilized by the purinergic receptor, we have started to examine the possibility that this pathway may interact during activation of Fin with the other two well-known receptor channel-coupling pathways present in follicular cells, adenylyl cyclase and phospholipase C (PLC) (3, 24). For these experiments, a pipette loaded with ATP (5-20 µM) was positioned near the oocyte's animal pole, and the pressure ejection was adjusted to obtain reproducible responses approximately every 60 s. The follicles were then superfused with FSH (0.5 µg/ml) to activate adenylyl cyclase in follicular cells and elicit adenosine 3',5'-cyclic monophosphate (cAMP)-dependent K+ currents (13, 33). All follicles tested (7 follicles, 3 frogs) behaved similarly, and the Fin in response to ATP did not change during the K+ current activated by the increase in cAMP synthesis (Fig. 3B). Note also that the cAMP-dependent K+ currents were not affected by brief ATP applications. FSH superfusion using saturating concentrations (2 µg/ml) or direct activation of the adenylyl cyclase pathway by forskolin (10 µM) were also ineffective on Fin (3 follicles, 2 frogs).

Interactions between membrane receptors that converge in activation of the PLC pathway in the oocyte are well known (24, 28). Thus, depending on concentration and order of application of two receptor agonists, one observes either potentiation or inhibition of ionic currents that use the same PLC-inositol phosphates-Ca2+ pathway. For example (cf., Fig. 16 in Ref. 24), sequential application of low concentrations of two agonists, which act on different receptors and activate the PLC pathway, regularly produces potentiation of the responses, whereas a saturating concentration of one agonist seems to transiently exhaust the system and inhibit the subsequent response to the same or different agonist. We therefore examined whether Fin activated by ATP were altered by costimulation of the PLC pathway by either ANG II (Fig. 3B) or ACh (Fig. 4B). Follicles with ANG II receptors located only in follicular cells (i.e., ANG II responses eliminated by defolliculation) were used preferentially for these experiments to activate the receptors and the PLC pathway only in that cellular compartment (30, 34). The Fin were activated by pulses of ATP from a pipette placed close to the follicle, and ANG II (100 nM) was superfused (8 follicles, 3 frogs) after a stable Fin amplitude was attained. As for the case of adenylyl cyclase activation, the Fin did not change during the superfusion of ANG II, which activated Ca2+-dependent oscillatory Cl- currents in the oocyte membrane, readily confirming activation of PLC and synthesis of inositol 1,4,5-trisphosphate (IP3) (5, 30, 34). In addition, in these same follicles, a full response produced by superfusion of ANG II saturating concentration (0.5-1 µM) did not inhibit the ATP-Fin responses, as we have previously reported (3).


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Fig. 4.   Interactions between Fin elicited by ATP and currents generated by ACh. A: cross-inhibition of follicular cell-based fast currents elicited by ACh on Fin generated by pulses of ATP (10 µM) in a follicle that responded to ACh with currents dependent on presence and coupling between follicular cells and oocyte. Apparent increase in onset delay of Fin elicited by ACh is due to dead time of perfusion system at a rate of ~2 ml/min. Same results were obtained in 12 follicles from 4 frogs. B: interaction between Fin evoked by pulses of ATP (10 µM) and oscillatory currents activated via muscarinic receptors located in oocyte membrane. Note lack of effect of activating PLC-pathway on ATP-Fin, particularly during generation of oscillatory current (ATP responses marked by arrowheads during oscillatory response). Typical results obtained in 5 follicles from 2 frogs. C: current responses elicited by ATP (50 µM) or ACh (50 µM), superfused at regular rate (10 ml/min), in follicles from same frogs used for records in A (a) and B (b), and in their respective oocytes that were collagenase treated and then manually defolliculated (c and d).

All these experiments strongly suggest that the ATP receptor channel pathway which activates the Fin does not converge potently with either the adenylyl cyclase or PLC pathway.

Applications of ATP pulses from a pipette placed close to the follicle, as those illustrated in Figs. 3B and 4, permitted us to also better measure the delay in activation of Fin (Fig. 3B, inset). When pipettes filled with ATP (5-100 µM) and pressure pulses of 100 ms (ejected drops were of ~200 µm in diameter, measured in air) were used, Fin had an onset delay of 460 ± 35 ms in 39 follicles from 5 frogs. The onset delay measured was not affected by the following changes: locating the micropipette to distances of 80-120 instead of 50 µm; increasing the rate of superfusion to 4-8 instead of 2 ml/min, although this reduced greatly the Fin amplitude; or increasing the ejection pulse duration from 100 to 200-1,000 ms, in which cases the amplitude and duration of Fin increased. Thus this seemed to indicate that the onset delay observed did not include notable components due to ATP diffusion from the ejection point to the follicular membrane. Furthermore and in sharp contrast with the oscillatory responses elicited by several agonists (3, 18, 24), it was also clear that the onset delay of the Fin response was not a function of the dose of ATP or of the current amplitude, suggesting a receptor channel mechanism different from that involved in the production of IP3 and subsequent opening of Cl- channels seen after activation of the PLC pathway in the oocyte.

Involvement of a G protein in the receptor channel-coupling mechanism was suggested by the result of experiments in which follicles were pretreated with pertussis toxin (1-10 µg/ml in Barth's medium). For these experiments, two groups of follicles were sequentially tested for FSH (0.5 µg/ml), ATP (50 µM), and ACh (50 µM). Responses to these agonists in a group of follicles (n = 13) that were pretreated with pertussis toxin (8-12 h) were compared with their controls (n = 15; 3 frogs). In control follicles, FSH elicited the typical K+ current activated through an increase of intracellular cAMP (13, 33), with an amplitude of 635 ± 285 nA, and ATP or ACh elicited Fin of 680 ± 250 and 856 ± 280 nA, respectively. In pertussis toxin-pretreated follicles, the FSH-elicited current was completely inhibited, whereas Fin activated by ATP or ACh were inhibited by 50% (355 ± 35 and 420 ± 120 nA, respectively). This suggests that the receptor channel-coupling mechanism in Fin activation is one that involves a pertussis toxin-sensitive G protein.

ATP-Fin interactions with follicle cholinergic responses. Like the ANG II receptors, ACh receptors can also be located in the membrane of the follicular cells and in the membrane of the oocyte itself (1, 17, 18). The apportioning of ACh receptors between the two compartments varies greatly among follicles from different frogs. Although a mixture is most common, some frogs have follicles with responses that originate only in the follicular cells or the oocyte (1, 3). Fin and Sin are typical responses caused by activation of ACh receptors located in the follicular cells, whereas activation of ACh receptors in the oocyte membrane elicits Ca2+-dependent Cl- currents with a characteristic oscillatory time course (Fig. 4C). In this study, we found that Fin, elicited by pressure ejection of ATP (10 µM in pipette), were cross-inhibited by the simultaneous application of ACh (25 µM) in follicles with ACh receptors located in the follicular cells (12 follicles, 4 frogs; Fig. 4A). In contrast, stimulation of ACh receptors located in the oocyte itself did not alter the Fin responses elicited by ATP (5 follicles, 2 frogs; Fig. 4B). For these two groups of follicles, the localization of the receptors was determined by defolliculation, which abolished Fin elicited by ATP or ACh but did not eliminate the ACh-oscillatory responses (Fig. 4C). These results again suggest an independence between Fin generation and PLC activation and also support our previous conclusion that the ACh receptors located in follicular cells activate a different set of Cl- channels from those activated in the oocyte membrane itself (1). It thus appears that the Cl- channels involved in the generation of ATP-Fin are the same as those involved in the generation of ACh-Fin and that most probably both follicular cell ACh and ATP receptors activate the same receptor-Cl- channel-coupling pathway.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

In this study, we show that most frogs yield follicles that respond to extracellular application of ATP by opening ionic Cl- membrane channels. Our results with a large sample of donors tested over several years indicate that frogs with follicles without responses to ATP are actually exceptional. Compared with previous reports (e.g., 16, 19, 20), careful dissection and culture conditions allowed us to maintain well the ATP currents. This resulted in follicular ionic currents that were ~10 times larger in amplitude and very well maintained for 6 days in cultured follicles. Because defolliculated oocytes did not show appreciable responses to ATP, our results confirm that Fin activated by ATP are follicular cell-based currents (3). We also show that these Fin are carried principally by Cl-, and there was no evidence of a significant permeability to Na+, as has been suggested before (16), or to K+. Moreover, Fin channels activated by ATP are permeable to other anions and are actually even more permeable to ions such as SCN-, I-, or Br-.

It seems clear that the purinergic receptor that activates the Fin is of the P2 type (3, 20), though one with an evidently atypical pharmacology. Our results show that ATP-Fin are fully mimicked by the purinergic receptor agonists UTP and 2-MeSATP, which activate inward currents that show potent cross-inhibition. As for ATP, the channels activated by UTP or 2-MeSATP are selectively permeable to Cl- with no significant permeability to Na+. Thus it is as if the follicular receptor is a P2 receptor that is sensitive to UTP, a P2U-like subtype; however, in contrast to the usual pharmacological definition (e.g., 9, 35), the follicular receptor has a strong sensitivity to 2-MeSATP and an order of potency in which 2-MeSATP > UTP > ATP. A possible explanation for this unusual order of potency is that P2Y receptors (activated by 2-MeSATP) and P2U receptors (activated by UTP) coexist in follicular cells activating the same set of Cl- channels and via the same membrane-coupling mechanism. This resembles the situation found for MDCK-D1 cells where P2Y and P2U receptors coexist, and stimulation of any of them produces accumulation of arachidonic acid and its metabolites (12). In bovine aortic endothelial cells, these two purinergic receptors subtypes also coexist (8, 10), and both are coupled to activation of PLC (8). Here, the inhibitory effects of suramin on Fin responses elicited by the agonists were differential, being more potent antagonist for ATP and 2-MeSATP than for UTP. This seems to support the existence of at least two different receptors in the membrane of the follicular cells. However, the possibility that the effects of purinergic agonists on follicular cells are due to the presence of a single type of purinergic receptor, with an unusual pharmacology, cannot yet be completely ruled out. An observation that supports the latter possibility is that the Fin activated by ATP, UTP, and 2-MeSATP are all of similar amplitude in follicles from a particular donor. This behavior is uncommon, compared with what is observed when one is dealing with activation of different follicular receptors. In that case, it has been shown that the potency of various agonists varies independently among different frogs (3, 24), as exemplified by our consistent observation of an independent ability of ATP and ACh in generating Fin in follicles from different frogs. It is also necessary to recall that suramin is a nonspecific antagonist of purinergic receptors. Several other effects of suramin have been reported on membrane proteins, particularly on G proteins (5) and other membrane receptors (4, 32). The results presented here are different from those reported in a previous study (16), in which fast (Fin) and slow (Sin) responses activated by ATP or UTP were similarly inhibited by suramin, whereas the slow response activated by 2-MeSATP was potentiated by the antagonist. The reasons for these different results remain unknown. Thus it seems that the effects of suramin on follicular currents are complex and may involve some other components of the activation pathway. It is clear that further pharmacological and molecular studies are required to determine whether one or more subtypes of purinergic and/or pyrimidergic receptors are present in the follicular cells.

ATP, UTP, and 2-MeSATP appear to activate Fin through a common membrane mechanism, as evidenced by the cross-inhibition experiments. These results also suggest that the inhibition occurs principally on the second messenger pathway activated or directly on the Fin Cl- channels, because the Fin elicited by ACh after activation of specific muscarinic receptors also had a potent inhibiting effect on the currents generated by ATP and vice versa.

Whatever the molecular nature of the receptor subtypes involved in the generation of follicular Fin turns out to be, the strongest candidates, the P2Y and P2U types, are both members of a large family of G protein-coupled membrane receptors that use predominantly inositol phosphates and Ca2+ signaling subsequent to stimulation of PLC, but purinergic receptors may also activate phospholipase A2 (12), stimulate phospholipase D (27), and promote protein tyrosine phosphorylation (29). Although the mechanisms involved in the generation of Fin by purinergic receptors in follicles are not yet fully understood, it seems that a G protein is involved. The results showed that pretreatment with pertussis toxin inhibited (~50%) the activation of Fin by both ATP and ACh. Also, the onset delay of Fin elicited by ATP was ~400 ms, whereas direct channel gating by nicotinic receptors in vertebrate skeletal muscle takes only a few microseconds (e.g., Ref. 15). We therefore anticipate that Fin channel activation by purinergic receptors involves an intermediary coupling membrane mechanism.

Here we present evidence for the notion that the receptor channel pathway involved in activation of Fin Cl- channels by purinergic receptors is not related to the synthesis of cAMP or to stimulation of PLC, two well-known messenger systems of follicular cells and oocytes. For instance, we show that Fin channel activity is not affected during ligand-induced oscillatory currents, which are carried through Ca2+-dependent Cl- channels or during K+ channel activation by an induced increase in cAMP. This is independent of whether the PLC is activated in the membrane of the follicular cells or in the oocyte itself, as is the case for ANG II or ACh, respectively, in this study. Furthermore, the Fin channels are not activated by a rise in intracellular Ca2+ concentration (1, 3), because intraoocyte injection of EGTA or BAPTA does not abolish the follicular responses. It still remains possible that these Ca2+ chelators, injected within the oocyte, did not reach sufficient concentration in the follicular cell compartment. Notwithstanding, it is clear that the Cl- channels involved in the Fin, elicited via purinergic or muscarinic receptors, are not the same as those mediating the Cl- currents that follow activation of PLC-coupled receptors of the oocyte membrane, because these currents are readily abolished by intraoocyte injection of Ca2+ chelators (1, 3, 22, 24).

The picture that emerges is that purinergic receptors mainly activate follicular cell-based currents, whereas receptors to ANG II, independent of their localization, activate oscillatory oocyte-based Cl- currents. Outward currents are occasionally elicited by ANG II, but these currents clearly originate in the follicular cells (Miledi and Arellano, unpublished results). In contrast, muscarinic receptors, which like ANG II receptors can be located in the follicular cells, the oocyte, or both compartments, can activate two types of inward current responses: one arising in the follicular cells and indistinguishable from the responses to ATP and the other the typical oscillatory current from the oocyte membrane. Follicles that have purinergic receptors in the follicular cells and muscarinic receptors only in the oocyte membrane (Fig. 4C) illustrate clearly the separation of these two types of responses. Nevertheless, generation of ancillary oscillatory currents by ATP or ACh in follicles from some donors and their elimination by defolliculation (Ref. 3; Miledi and Arellano, unpublished results) suggest a weak coupling of the PLC pathway to the muscarinic and purinergic receptors involved in generating the Fin.

Finally, we found that short pulses of ATP did not inhibit K+ currents elicited by FSH (or forskolin), even though the Fin were activated. In contrast, cAMP-K+ currents are almost completely inhibited by superfusion of ACh (31, 33) or ATP (3). Therefore, if activation of Fin and inhibition of cAMP-K+ currents are elicited via the same purinergic receptor, our results suggest that inhibition is a late step in the reaction cascade triggered by ATP. It is still possible that the pathway activated by ATP interacts with the cAMP system, but this will necessarily be subsequent to generation of the Fin.

Purinergic receptors have been described in follicular cells (granulosa cells) from other animal species (e.g., 14, 26). Therefore, results presented here, coupled with further studies of the mechanisms activated by purinergic receptors in Xenopus follicles, may help to better understand the functions of ATP in other ovarian systems. As more pharmacological and molecular information becomes available, this will also help to define possible relationships between members of the purinergic receptor family.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Edgar Heimer for help with the manuscript.

    FOOTNOTES

This work was supported by grants from The Third World Academy of Sciences (95-502 RG/BIO/LA) and UNAM-DGAPA (IN209596) to R. O. Arellano and National Institute of Neurological Disorders and Stroke Grant NS-23284 to R. Miledi.

Address for reprint requests: R. O. Arellano, Centro de Neurobiología, Universidad Nacional Autónoma de México, Apartado Postal 1-1141, Queretaro, QRO, CP 76001, Mexico.

Received 25 June 1997; accepted in final form 17 October 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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