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1 Institut National de la Santé et de la Recherche Médicale, Unité 426, Faculté de Médecine Xavier Bichat, 75870 Paris Cedex 18; and 2 Laboratoire de Neurophysiologie Sensorielle, Université de Rouen, 76821 Mont-Saint-Aignan Cedex, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study was
attempted to characterize pharmacologically the P2Y receptors
triggering phospholipase A2 (PLA2) activation in ampulla from frog semicircular canal. A microassay was developed to
screen the abilities of UTP analogs to stimulate
[3H]arachidonic acid release by labeled ampullas. At
26°C UTP induced a dose-dependent and saturable increase of
PLA2 activity (apparent activation constant 1.3 ± 0.4 µM, Hill coefficient 0.9 ± 0.2, maximal stimulating factor
2.0 ± 0.1). The rank order of potency of agonists for
PLA2 activation was UTP 
UDP > adenosine
5'-O-(2-thiodiphosphate) = adenosine
5'-O-(3-thiotriphosphate) ATP = 2-methylthio-ATP ADP = diadenosine tetraphosphate 
,
-methylene-ATP = CTP > 2' and
3'-O-(4-benzoylbenzoyl)-ATP AMP = UMP >>
uridine and adenosine. UTP- and 2-methylthio-ATP-induced
PLA2 activations were inhibited by U-73122, GF-109203X, and
methyl arachidonyl fluorophosphate. Basal activity was stimulated by
phorbol ester and epinephrine and reduced by vasotocin, isoproterenol,
prostaglandin E2, cAMP, and forskolin. H-89 restored
the cAMP- and forskolin-inhibited PLA2 activities. Results
indicate that P2Y receptor-mediated PLA2 stimulation
requires phopholipase C and protein kinase C activations and basal
activity is inhibited by agonist-stimulated cAMP-dependent mechanisms.
inner ear; arachidonic acid release; P2Y receptors; UTP analogs
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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EXTENSIVE FINDINGS PERFORMED on cultured cell lines have clearly established that extracellular triphosphonucleotides stimulate phospholipase A2 (PLA2) activity, leading to increases in arachidonic acid (AA) release and subsequent synthesis and secretion of prostaglandins through activation of cyclooxygenases (9, 15, 22, 25, 33).
For Tetrapod inner ear organs, evidence has been provided for regulating roles of extracellular ATP and UTP on neurotransmission and endolymph homeostasis (1, 16, 29). ATP was found in perilymphatic and endolymphatic fluids of the cochlea (21), and it was reported that prostaglandins are released by Rana esculenta semicircular canal (8).
Biochemically, two types of P2 purinoceptors functionally coupled to phospholipase C (PLC) activation have been characterized in ampulla from R. ridibunda semicircular canal (3, 4, 28). On the basis of their rank orders of potencies for recognition of ATP structural analogs, these receptors resembled to mammalian P2Y1 and P2(Y) receptors (11) and were called P2Y1-like and P2(Y)-like receptors (4, 28). In the gerbil vestibule and cochlea, it was assumed that the UTP-induced inhibition of the vectorial transport of K+ toward the endolymphatic compartment is mediated by PLC stimulation followed by protein kinase C (PKC) activation through diacylglycerol-dependent and inositol trisphosphate- and Ca2+-independent mechanisms (16-18).
The aim of the present work was to screen, using a series of structural UTP analogs more selective for the P2Y receptors linked to G protein signal pathways or P2X ligand-gated ion channels (10, 11), the pharmacological properties of the P2 receptors triggering PLA2 stimulation in isolated ampullas microdissected from R. ridibunda semicircular canals. Our results indicate that the nucleotide-induced PLA2 activation requires occupancy of P2Y1-like and/or P2(Y)-like receptors (11), and PLC and PKC stimulation. Basal PLA2 activity is inhibited by agonists acting through cAMP-dependent pathways.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Products Used
Arachidonic acid [5,6,8,9,11,12,14,15-3H(N)] ([3H]AA; 8.03 TBq/mmol) was provided by New England Nuclear (Le Blanc Mesnil, France).Other chemicals were purchased from the following
sources: adenosine, AMP, cAMP, 8-bromoadenosine 3',5'-cyclic
monophosphate (8-BrcAMP), ADP, adenosine
5'-O-(2-thiodiphosphate) (ADPS), ATP, adenosine
5'-O-(3-thiotriphosphate) (ATP
S),
,-methyleneadenosine 5'-triphosphate (,-Me-ATP), 2'- and
3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (Bz-ATP),
CTP, diadenosine tetraphosphate (Ap4A), uridine, UMP, UDP,
UTP, arginine vasotocin (AVT), bradykinin, epinephrine, isoproterenol,
phenylephrine, phorbol 12-myristate 13-acetate (PMA), prostaglandin
E2 (PGE2),
N-[2-(p-bromocinnamylamino)-ethyl]-5-isoquinolinesulfonamide (H-89), DIDS, reactive blue 2 (basilen blue E-3G), bacitracin, BSA, and
forskolin from Sigma (St. Louis, MO); 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) and
pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) from
Research Biochemicals International (Natick, MA); bisindolylmaleimide I
(GF-109203X), methyl arachidonyl fluorophosphate (MAFP), and
1-{6-[(17-3-methoxyestra)-1,3,5(10)- (trien-17-yl)amino]hexyl}-1H-pyrrole-2,5-dione (U-73122) from Calbiochem (Meudon, France); suramin from Miles (Naperville, IL).
Animals
Experiments were performed using adult frogs of both sexes of the species R. ridibunda purchased from the Contigné Breeding Center (France). Frogs were kept in tanks containing tap water at 8°C. They were decapitated. The head was split in two halves and the brain was destroyed (approval of the Ministère de l'Agriculture et de la Pêche, No. 5521).Microdissection of Ampullas From Frog Semicircular Canals
Microdissection of the three semicircular canals from each inner ear was performed at 4°C under stereomicroscopic observation in a modified amphibian Ringer solution, medium A (in mM, 20 HEPES-NaOH, pH 7.5, 82 NaCl, 3 KCl, 1.8 CaCl2, 1 MgCl2, 0.33 NaH2PO4, 0.44 Na2HPO4, 5 glucose, and 10 sodium acetate, and 0.1% BSA). The ampullas were separated from the adjacent regions of the semicircular canals and opened by saggital incision of their dorsal aspects. In some experiment, the dorsal ampullary region formed of undifferentiated epithelial cells was separated from the ventral region containing secretory dark cells, transitional cells, sensory hair cells, and undifferentiated cells (23) by cutting the higher frontal level of ampulla. Secretory dark cell and sensory hair cell areas with their adjacent connective tissue were microdissected from other structures in the ventral ampullary region. Epithelial structures were kept at 4°C until use.PLA2 Studies
[3H]AA release. Assays were performed using a microtechnique adapted to frog ampullas derived from the method described for cultured cell lines (9, 24). Ampullas were labeled in 40 µl of medium B (medium A containing 18.5 kBq [3H]AA) for 3 h at 27.5°C in a humid atmosphere and then extensively washed in a large volume of medium A at room temperature to eliminate extracellular [3H]AA.
One ampulla was used for each individual determination. The incubation was started by transferring ampulla onto a 10-µl droplet of medium C (medium A containing 25 mM HEPES-NaOH, pH 7.5, 0.5% BSA, 0.1% bacitracin, various amounts of UTP or structural analogs, agents of interest or vehicles) put on the hollow of a siliconized bacteriological slide, and the sample was tightly covered with a petroleum jelly-coated slide to obtain a water-tight seal. The reaction was carried out for 30 min at 26°C and stopped by adding 200 µl of chilled medium D (medium C plus 5 mM EGTA-NaOH, pH 7.5, and 5 mM EDTA-NaOH, pH 7.5). The ampulla was removed, treated with 10 µl NaOH 1 N, and counted for 10 min for determination of tissular-associated radioactivity. The medium was recovered and counted twice for 20 min for measurement of released [3H]AA and derived 3H-labeled metabolites. Generally for each experiment, six to eight concentrations of a given UTP analog were tested in six replicates. Therefore, each dose-response curve was drawn from data obtained with 36-48 ampullas microdissected from six to eight frogs.Calculations.
Results were expressed as ratios between released radioactivity and
total radioactivity incorporated (the latter was 3,437 ± 242 counts · min
1 · ampulla1;
means ± SE of mean data from 42 experiments with 30-64
individual determinations).
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max) and of UTP (max UTP)
measured in the same experiment.
Control experiments. It was checked that 1) both basal and 0.1 mM UTP-stimulated enzyme activities increased linearly with incubation time up to 30 min at 26°C; 2) at the end of the PLA2 assay, a second washing of ampullas with medium D at 4°C did not induce supplementary release of [3H]AA and [3H]-labeled-derived metabolites. Basal and 50 µM UTP-stimulated PLA2 activities (%total radioactivity incorporated; means ± SE of 6 replicates) of control samples were 4.2 ± 0.4 and 7.9 ± 0.7 and those of washed samples were 4.5 ± 0.6 and 8.1 ± 0.8, respectively (differences between corresponding basal or UTP-stimulated activities are not significant; Student's t-test); and 3) the presence of 2.5% dimethyl sulfoxide (DMSO) in the incubate enhanced basal PLA2 activity. The latter were (%total radioactivity incorporated; means ± SE of mean data from number of experiments) 5.7 ± 0.7, N = 6, and 3.9 ± 0.2, N = 36 for DMSO-treated and control samples, respectively (P < 0.025, Student's t-test).
Statistical Analysis
Results were given as means ± SE of n (replicates performed in the same experiment) or as means ± SE of mean data from N (different experiments). When appropriate, differences were analyzed using Student's t-test; the latter were considered significant at P < 0.05.For each analog tested, the 95% confidence interval variation range of
pKa value (pKa = 
log Ka, in which Ka was
expressed as molar concentration) for nucleotide-induced
PLA2 activation was calculated by computerized analysis of
the corresponding dose-response curves (GraphPad Prism Software;
sigmoidal dose-response fit with variable slope). It was assumed at the
95% probability level that two analogs stimulate PLA2 with
differing apparent affinities, only when the lower limit of
pKa variation range of the best nucleotide is
higher than the upper limit of pKa variation
range of the worst analog.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Figure 1 shows that incubation of
[3H]AA-labeled ampullas with UTP increased the basal
release of [3H]AA and derived 3H-labeled
metabolites. The UTP-induced PLA2 activation was
dose-dependent, saturable with nucleotide concentration, and
characterized by the following parameters (means ± SE of
N experiments): 1) a threshold response for about
0.1 µM UTP; 2) an apparent activation constant Ka = 1.3 ± 0.4 µM,
N = 6; 3) a Hill coefficient
nH = 0.86 ± 0.15, N = 6;
4) a maximal response observed for about 50 µM UTP
(maximal stimulating factor = 1.98 ± 0.09, N = 23); and 5) self-inhibitory effects for nucleotide
concentrations higher than 50 µM.
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The sensitivity of PLA2 of ampulla from frog semicircular
canal to structural UTP analogs is illustrated by typical experiments depicted in Fig. 2, and all results are
summarized in Table 1. It is worth noting
that the nucleotides tested were able to stimulate [3H]AA
release with differing potencies as regards 1) their
apparent activation constants, the Ka values of
the P2(Y) agonists UTP and UDP were smaller than those of the
long-acting analogs ADPS and ATPS; those of the natural
nucleotides ATP, ADP, and CTP; and those of the potent markers of the
P2Y1 receptors (2-MeS-ATP), P2YAp4A receptors
[Ap4A (11)], and P2X1 and
P2X3 receptors (,-Me-ATP), respectively. They were
also far lower than that of the selective P2X7 agonist
(Bz-ATP) and those of AMP and UMP; 2) their Hill coefficients (ADPS, ATPS, Ap4A, and ,-Me-ATP
stimulated enzyme activity with negative cooperativity phenomena, UTP,
UDP, ATP, ADP, CTP, and Bz-ATP according to Michaelian kinetics and
2-MeS-ATP with slight positive cooperativity phenomena); and
3) their intrinsic activities or magnitude of the maximal
responses observed (ATP was about 1.6 times more potent than UTP;
2-MeS-ATP, Ap4A, CTP, Bz-ATP, AMP, and UMP exhibited
partial agonistic potencies, whereas 10 mM of uridine or adenosine were
inactive).
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Data from a competitive experiment between the potent P2(Y) agonist UTP
and the selective P2Y1 agonist 2-MeS-ATP (10,
11) clearly indicate that the responses to saturating amounts of
both nucleotides were not additive. The increases in
[3H]AA release (, %total radioactivity incorporated;
means ± SE of 8 replicates) above basal activity (2.2 ± 0.2) induced by 50 µM UTP, 0.3 mM 2-MeS-ATP, and 50 µM UTP + 0.3 mM 2-MeS-ATP were, respectively, 4.2 ± 0.6, 2.8 ± 0.6, and 4.7 ± 0.4; the latter value is significantly lower than the
sum of both activations (computed , 7.0 ± 0.8;
P < 0.01; Student's t-test). These data demonstrate that UTP and 2-MeS-ATP stimulate the same pool of PLA2.
As expected, the unrelated nucleotide chemicals revealing antagonistic
properties (3, 4, 10, 11, 28) were able to inhibit
UTP-induced PLA2 activation (Table
2). In the presence of antagonist
concentrations leading to total inhibition of specific UTP binding
(28) (5 mM DIDS, 2 mM reactive blue 2, 10 mM PPADS, and 10 mM suramin), the basal enzyme activity was decreased by about 50% by
DIDS and not significantly impaired by reactive blue 2, PPADS, or
suramin, whereas the activation of [3H]AA release
elicited by 1 µM UTP vanished in the presence of the four antagonists
tested.
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Results depicted in Fig. 3 show the
effects of inhibitors of either PLC [U-73122 (12)], PKC
[GF-109203X (31)], or cytosolic PLA2 [MAFP
(19)] on PLA2 activity. The presence in the
assay medium of 50 µM U-73122, 10 µM GF-109203X, or 50 µM MAFP
did not alter basal [3H]AA release but decreased by about
50% the PLA2 activations induced by maximal amounts of UTP
or 2-MeS-ATP. These observations suggest that P2Y-like
receptor-mediated stimulation of [3H]AA release
requires PLC and PKC activations.
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It was checked that the basal release of [3H]AA was
stimulated about 1.3, 1.4, and 1.8 times by 0.1 mM phenylephrine, 0.1 µM PMA, and 0.1 mM epinephrine, respectively, but was not modified by
10 µM bradykinin. Interestingly, it was found that 10 µM AVT, 10 µM isoproterenol, 0.1 µM PGE2, or 10 mM cAMP reduced
basal enzyme activity by 21, 31, 49, and 42%, respectively
(P < 0.05, Student's t-test). Moreover,
results illustrated in Fig. 4 demonstrate that these inhibitory effects were reproduced by 1 mM of the permeant cAMP analog 8-BrcAMP or by 5 µM forskolin that decreased
significantly basal PLA2 activity by about 35%. The
8-BrcAMP- and forskolin-induced inhibitions of basal
[3H]AA release vanished in the presence of 50 µM of the
protein kinase A (PKA) inhibitor [H-89 (32)]. In
addition, enzyme responses to saturating amounts of UTP or 2-MeS-ATP
were not impaired by 1 mM 8-BrcAMP or 5 µM forskolin (data not
shown).
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Finally, the distribution of nucleotide-sensitive PLA2
activities in the different regions of ampulla from frog semicircular canal is presented in Table 3. Results
show clearly that UTP and 2-MeS-ATP stimulated [3H]AA
release in the dorsal region, the ventral region, and in the dark cell
areas, whereas neither agonist enhanced basal activity in the hair cell
area.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The experiments described provide evidence for the expression of a nucleotide-sensitive PLA2 activity in ampulla of R. ridibunda semicircular canal. Data also show that basal release of [3H]AA and derived 3H-labeled metabolites was stimulated by the selective P2(Y) receptor agonist (UTP) and the potent P2Y1 receptor marker (2-MeS-ATP) (10, 11) in the dorsal region, the ventral region, and in the dark cell areas but not in the hair cell area of the ampullary epithelium.
Despite the minute amounts of tissue used [about 2.5 µg total protein/whole ampulla (28)], the validity of the microassay employed was verified by the following experiments: 1) basal and UTP-stimulated releases of [3H]AA and derived 3H-labeled metabolites increased linearly with incubation time up to 30 min; 2) agonist-induced PLA2 activations were dose dependent and saturable; 3) purinoceptor antagonists inhibited UTP-stimulated enzyme activity; and 4) the selective cytosolic PLA2 inhibitor MAFP decreased the activations induced by maximal concentrations of UTP and 2-MeS-ATP.
It seems unlikely that the differences found between Ka values of agonists for PLA2 stimulation result from ecto-ATPase and ecto-nucleotidase activities (5, 6, 30) because it was reported earlier that a triphosphonucleotide-regenerating system did not impair basal, submaximal, and maximal ATP- and UTP-stimulated PLC activities in frog ampulla (3, 28).
It must be pointed out that data from a competition experiment between UTP and 2-MeS-ATP show that the PLA2 responses to saturating amounts of both agonists were not additive. This observation indicates that occupancies of P2(Y)-like receptors by UTP and of P2Y1-like receptors by 2-MeS-ATP lead to stimulation of the same pool of PLA2 in frog ampulla.
Results of pharmacological investigations demonstrate that
nucleotide-induced PLA2 activation is mediated by
P2Y1-like and/or P2(Y)-like receptor occupancy. Indeed
1) the potent marker of P1 receptors (adenosine) was
inactive and the selective ligands for P2X1 and
P2X3 receptors (,-Me-ATP) and for P2X7
receptors (Bz-ATP) stimulated PLA2 with only low affinities
and 2) the antagonists reactive blue 2, DIDS, PPADS, and
suramin that inhibit competitively UTP binding and UTP-induced PLC
activation in frog ampulla (28) abolished UTP-stimulated
PLA2 activity.
On the one hand, both the occurrence of a close linear relationship
between the pKa values of 11 potent structural
UTP analogs for PLA2 activation and their corresponding
values for PLC stimulation (Fig. 5) and
the observed decreases of UTP- and 2-MeS-ATP-induced PLA2
activations in the presence of the selective PLC inhibitor U-73122,
suggest strongly that PLC activation is involved in the purine- and
pyrimidine-dependent mechanisms triggering PLA2
stimulation. In the other systems so far studied, it was reported that
PLC activates PLA2 via PKC (9, 15, 22, 25,
33). In frog ampulla, a similar pathway may be suggested on the
basis of the following observations: 1) the PKC activator
PMA increased basal PLA2 activity and 2) the PKC
inhibitor GF-109203X reduced UTP- and 2-MeS-ATP-induced
PLA2 activations.
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On the other hand, it should be stressed that cAMP and forskolin, as
well as AVT, isoproterenol, and PGE2 that enhance
intracellular cAMP levels in frog ampulla (7) were able to
decrease basal PLA2 activity. These agonist-induced
inhibitory effects are likely mediated by intracellular cAMP-dependent
pathways rather than by occupancy of P2Y-like receptors by the
extracellular cAMP released from intracellular stores because
1) cAMP did not interact with [35S]ADPS-
and [3H]UTP-labeled binding sites and did not impair
basal PLC activity in frog ampulla (3, 4, 28) and
2) the PKA inhibitor H-89 restored to control level the
8-BrcAMP- and forskolin-induced inhibitions of basal PLA2
activity. This latter observation suggests that PKA activation is
implicated in the cAMP-dependent mechanisms triggering PLA2 inhibition.
On these grounds, it might be assumed that the nucleotide-dependent
PLA2 stimulation and cAMP-dependent enzyme inhibition involve the following biochemical steps: 1) occupancy by
extracellular purines and pyrimidines of the P2Y1-like
and/or P2(Y)-like receptors coupled to PLC activation triggering direct
activation of PKC through diacylglycerol-dependent mechanisms as
observed in the gerbil vestibule and cochlea (17, 18);
2) phosphorylation of cytosolic PLA2 by PKC
leading to increases in AA release and subsequent synthesis and
secretion of prostaglandins through cyclooxygenase activation as
reported in Madin-Darby canine kidney (MDCK) cells (25,
31); 3) PGE2 receptor-mediated adenylate
cyclase activation rising intracellular cAMP levels as shown in frog
semicircular canal and MDCK cells (7, 25); and
4) cAMP-stimulated PKA activity inhibiting cytosolic
PLA2 activity as described in MDCK cells
(32). So the stimulation of AA release and
prostaglandin synthesis triggered by purine P2Y1-like
and/or pyrimidine P2(Y)-like receptor occupancy could be regulated by a
negative feedback loop (Fig. 6). Thus it
will be interesting to further investigate in frog semicircular canal,
the effects of extracellular nucleotides on prostaglandin release,
intracellular cAMP levels, and adenylate cyclase sensitivity to AVT,
isoproterenol, and PGE2.
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Perspectives
The expression in frog ampulla of P2Y1-like and P2(Y)-like receptors involved through PLC- and PKC-dependent mechanisms in the regulation of PLA2 activity and likely in the synthesis and release of prostaglandins raises obvious questions about their cellular localization(s) and physiological function(s). Purine- and pyrimidine-sensitive PLA2 activities were found mainly in the undifferentiated cells of the dorsal region and in the secretory dark cell areas but not in the sensory hair cell area of the ventral region, whereas ATP receptors and purine-sensitive PLC activities have been characterized in all structures of the frog ampulla (3, 4).These observations suggest that the P2Y receptors borne by ampullary hair cells trigger prostaglandin-independent biological effects and strengthen data suggesting that hair cells produce low amounts of prostaglandins. Indeed, streptomycin treatment of ampullas that destroys sensory cells, did not alter significantly prostaglandin synthesis (8). Moreover, in sensory structures of the inner ear, ATP increased the spontaneous electrical activity of afferent fibers from R. pipiens semicircular canal and Xenopus laevis lateral line (1, 20), it induced lysis of outer hair cells, and altered gross cochlea, auditory nerve potential, and distorsion product otoacoustic emissions in guinea pig cochlea (2, 13, 14, 29). Therefore, the molecular subtype(s) and physiological significance(s) of the P2Y receptors expressed in Amphibia sensory cells call for additional electrophysiological, biochemical, and molecular cloning experiments.
Finally, the presence of a purine- and pyrimidine-sensitive PLA2 activity in the dorsal region and in the dark cell areas of the ventral region of frog ampulla might reflect an ubiquitous localization of P2Y1-like and P2(Y)-like receptors mediating AA and prostaglandin releases as reported for many other systems (9, 15, 22, 25, 33). This nucleotide-dependent PLA2 activity also might be involved in the functions of secretory dark cells. Indeed, it was proposed that prostaglandins could be implicated in the regulation of endolymphatic fluid composition (26), and it was reported that both ATP and UTP decrease the vectorial transport of K+ toward the endolymphatic space in the gerbil vestibule and cochlea, wheras cAMP increases K+ secretion via activation of IsK/KvLQT1 channels in strial marginal cells (16, 27). Thus further electrophysiological experiments will be necessary to investigate the effects of purine and pyrimidine nucleotides, cAMP, and prostaglandins on the electrogenic K+ secretion by ampulla from frog semicircular canal.
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ACKNOWLEDGEMENTS |
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We are indebted to Prof. Gérard Friedlander for critical advice and stimulating discussions.
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FOOTNOTES |
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This work was supported by grants from Institut National de la Santé et de la Recherche Médicale and Université Paris 7.
Address for reprint requests and other correspondence: D. Butlen, INSERM U. 426, Faculté de Médecine Xavier Bichat, 16, rue Henri Huchard, BP 416, 75870 Paris Cedex 18, France (E-mail: u426{at}bichat.inserm.fr).
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 26 May 2000; accepted in final form 20 September 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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