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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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(
-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 -methylene ATP (
-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).
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RESULTS |
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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
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Fin responses elicited
by purinergic agonists.
From a group of purinergic agonists (ADP, AMP, and
-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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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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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.
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Edgar Heimer for help with the manuscript.
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FOOTNOTES |
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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.
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