1 Department of Physiology and Pediatrics and 2 Division of Gastroenterology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We investigated the process of and recovery from desensitization
of the P2 receptor-mediated
stimulation of Cl secretion
in Madin-Darby canine kidney (MDCK) cell monolayers by assaying the
response of short-circuit current
(Isc). When the
cells were exposed to repeated 3-min challenges of ATP or UTP
interspersed with 5-min washes, the response of
Isc desensitized rapidly followed by spontaneous recovery. The pattern of inhibition by
various channel blockers or enzyme inhibitors revealed that both the
initial and recovered responses of
Isc have the same ionic and signaling mechanisms. The desensitization and recovery processes were confined to the membrane exposed to the repeated challenges. When added during the desensitized phase, 8-bromoadenosine 3',5'-cyclic monophosphate enhanced the ATP-stimulated
Isc response, whereas it did not during the initial or recovered phases. ATP-induced increases of intracellular adenosine 3',5'-cyclic
monophosphate showed similar desensitization and recovery in parallel
with the changes in the responses of
Isc. The
desensitization process was attenuated by pretreatment with cholera
toxin or pertussis toxin. Taken together, our results suggest that the
adenylyl cyclase system plays a role in the desensitization and
recovery mechanism of the ATP-stimulated
Cl
secretion in MDCK cells.
P2 receptor; short-circuit current; adenosine 3',5'-cyclic monophosphate; cholera toxin; pertussis toxin
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
EXTRACELLULAR ATP ACTS as an agonist to regulate a
broad range of physiological processes by interacting with the
P2 receptors, which can be grouped
into two subfamilies (for reviews, refer to Refs. 2, 7, 10, 11, 15).
The P2x receptors are ligand-gated
cation channels with a higher affinity to ,
- or
,
-methylene
ATP. The G protein-coupled receptor superfamily includes the
P2y (or
P2y1),
P2u (or
P2y2), and
P2t (or
P2y3) receptors. Among them, the
P2t subtype, which is activated by
ADP, is expressed in a very limited number of cell types. The
P2y and
P2u receptors are widely expressed
in various tissues. The latter can be discriminated pharmacologically
by the agonist selectivity: 2-methylthio-ATP (2-MeSATP) > ATP > ADP
>> UTP for P2y and UTP = ATP > adenosine 5'-O-(3-thiotriphosphate)
(ATP
S) >> 2-MeSATP = ADP for
P2u.
Desensitization of the G protein-linked receptor-mediated responses is a widespread phenomenon occurring upon prolonged or repeated exposure(s) to agonists (24). The P2 receptors show both heterologous and homologous desensitization (33). The molecular events involved in the desensitization are not entirely clear, but it has been suggested that the phospholipase C (PLC) products, Ca2+ or protein kinase C, act as negative-feedback modulators of receptor-G protein or G protein-PLC interaction (6, 8, 25).
In epithelial cells found in the kidney (29), airway (18, 20), and
intestine (9), ATP stimulates
Cl secretion through
multiple purinoceptors and signaling mechanisms. G protein-dependent
activation of PLC and subsequent increase in intracellular
Ca2+ is a major signaling
mechanism that stimulates
Cl
secretion. Madin-Darby
canine kidney (MDCK) cells are a well-differentiated cell line and have
many characteristics of the distal nephron (14, 32). They are widely
used as a model system for studying the regulation of epithelial cell
function. They are known to have an ATP-stimulated
Cl
secretory mechanism
(28). Recently, Post et al. (23) demonstrated that the
P2 purinergic agonists enhance
adenosine 3',5'-cyclic monophosphate (cAMP) production in
these cells.
In the course of preliminary experiments on ATP-regulated
Cl secretion in MDCK cells,
we also observed the desensitization of the ATP-stimulated increases in
short-circuit current
(Isc), but with
an interesting and unexpected phenomenon, spontaneous and gradual
recovery from desensitization with repeated challenges. This initial
result prompted us to investigate further the mechanisms of the
desensitization and recovery of the ATP-stimulated
Cl
secretion in MDCK cells.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
ATP, ATPS, UTP,
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS),
amiloride, quinine, indomethacin, and bumetanide were purchased from
Sigma Chemical (St. Louis, MO). 2-MeSATP and adenosine were
obtained from Research Biochemicals International (Natick,
MA). Forskolin, 8-bromoadenosine 3',5'-cyclic
monophosphate (8-BrcAMP),
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), cholera toxin, pertussis toxin, and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) acetoxymethyl ester (AM) were obtained from Calbiochem (La
Jolla, CA). Diphenylamine-2-carboxylate (DPC) was from Fluka Chemie
(Buchs, Switzerland). Eagle's minimal essential medium (MEM) and fetal
bovine serum (FBS) were purchased from GIBCO (Grand Island, NY).
Cell culture. MDCK cells obtained from American Type Culture Collection were routinely maintained on plastic culture flasks in MEM supplemented with 10% FBS, 50 IU/ml penicillin G, and 50 µg/ml streptomycin. Cells were trypsinized when cells became confluent (approximately every 4-5 days) using 0.05% trypsin-0.53 mM EDTA solution and reseeded at one-sixth the original density. For Isc measurement, cells were subcultured at a density of 5 × 105 cells on 12-mm polycarbonate membrane filters (Snapwell, Costar, Cambridge, MA). The cells were fed with fresh media every other day and the day before the experiments. The cells used for this study were between passages 86 and 110.
Isc measurements. Isc measurement was performed in a modified Ussing chamber designed to accept Snapwell filters (World Precision Instruments, Sarasota, FL). The transepithelial potential difference was short circuited with a voltage clamper (model DVC-1000, World Precision Instruments) connected to apical and basolateral chambers via Ag/AgCl electrodes. The experiments were carried out in bicarbonate-free Ringer solution that was composed of (in mM) 140 NaCl, 2.3 K2HPO4, 0.4 KH2PO4, 1.5 CaCl2, 1.5 MgCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 5 glucose (pH 7.4). Both the apical and basolateral bathing solutions were maintained at 37°C, oxygenated with 100% O2, and subject to constant circulation. Before the stimulation by agonists, the cell monolayers were equilibrated in Ringer solution for 30 min.
Measurement of intracellular cAMP content. Cells were grown on Snapwell filters and subject to the same procedure as in measurement of Isc. After 3-min exposure to the agonists, the Snapwells were rapidly removed from the Ussing chambers and immersed in ice-cold ethanol HCl (ethanol solution containing 20 mM HCl). The membrane filters were cut off the Snapwell support using sharp tuberculin needles. Cells attached to the membrane filters in ethanol HCl solution were then transferred to microcentrifuge tubes and sonicated to disrupt the cell membrane and complete extraction of intracellular cAMP. The cell suspension was then centrifuged (12,000 g) for 10 min at 4°C to precipitate the protein, and the supernatant was collected. The supernatant was freeze-dried and dissolved in an adequate volume of 50 mM tris(hydroxymethyl)aminomethane/1 mM EDTA (pH 7.5). cAMP content was determined by radioimmunoassay using [3H]cAMP assay kit from Amersham (Arlington Heights, IL). Protein concentration was determined using the Bio-Rad protein assay kit with bovine serum albumin as standard.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Desensitization and recovery of ATP stimulation of
Isc .
Figure 1 shows a typical response of
Isc in MDCK cells
exposed to repeated doses of 10 and 100 µM ATP added into the apical bathing solution. The protocol involved repeated 3-min exposures punctuated by a 5-min wash period. During the wash period, previously added ATP was carefully washed out by irrigation with Ringer solution four or five times so that it did not affect the integrity of tight
junctions. With this maneuver, the
Isc and
transepithelial resistance returned to the basal level usually 2-3
min before the next application of ATP. The basal
Isc and
transepithelial resistance in the MDCK cell monolayer used for this
study were in the range from 0 to 4 µA (mean = 2.7 µA) and 2,450 to
5,230 · cm2
(mean = 4,310
· cm2),
respectively.
|
Effect of various channel blockers or enzyme inhibitors.
The pattern of inhibition by various channel blockers or enzyme
inhibitors in Fig. 2 showed that the same
ionic and signaling mechanisms are involved in the ATP-stimulated
Isc responses in both the initial and recovered phases. Although we did not present the
result here, when the Cl in
the experimental solution was replaced with gluconate, the ATP-stimulated
Isc response
disappeared almost completely. This result, together with the
significant inhibition by bumetanide in the basolateral bathing
solution (Fig. 2), suggests that the ATP-stimulated
Isc responses
during both the initial and recovered phases can be ascribed to
basolateral to apical secretion of
Cl
. Both the initial and
recovered responses of
Isc to ATP were significantly inhibited by the
Cl
channel blocker (1) DPC
(200 µM, apical side). In contrast, DIDS (200 µM, apical side),
which is known to block some
Ca2+-activated
Cl
channels (5), or
glibenclamide (200 µM, apical side), which blocks cystic fibrosis
transmembrane conductance regulator (CFTR) (27), was without effect.
This result suggests that the
Ca2+-activated
Cl
channel or CFTR is not
likely to be involved in the apical
Cl
conductance. However,
significant inhibition by the protein kinase A inhibitor (4) H-89 (20 µM, both sides) suggests that the stimulation of
Cl
conductance is dependent
on protein kinase A. The inhibition by the intracellular
Ca2+ chelator (16) BAPTA-AM (10 µM, both sides) and the nonspecific K+ channel blocker (30) quinine
(500 µM, basolateral side) suggests that activation of basolateral
Ca2+-dependent
K+ channels is also very important
to the ATP-induced stimulation of
Isc. The
ATP-stimulated
Isc response was
inhibited almost completely by indomethacin (10 µM, both sides). This
result strongly suggests that the metabolites of arachidonic acid are
involved in the signaling mechanism.
|
Responses to apical vs. basolateral stimulation. A similar pattern of desensitization followed by recovery occurred with exposures to ATP in the basolateral bathing solution (Fig. 3, top right). To address whether this phenomenon occurs in the exposed membrane only, we determined whether the responses to ATP in the contralateral side are affected during the desensitization and recovery phases induced by repeated exposures to ATP in the apical or basolateral side. Figure 3 shows the typical responses when the epithelium is exposed repeatedly to apical ATP (top left). During the initial, desensitized, and recovery phases of the response to apical application, a single dose of ATP was applied to the basolateral cell membrane (Fig. 3, bottom left). The results show that repeated application of ATP to the apical solution does not affect the response to basolateral application. Figure 3 also shows the typical response when the epithelium is exposed repeatedly to basolateral ATP (top right). During the initial, desensitized, and recovery phases of the response to basolateral application, a single dose of ATP was applied to the apical membrane (Fig. 3, bottom right). The results show that repeated application of ATP to the basolateral solution does not affect the response to apical application. These results demonstrate that the desensitization and recovery process is confined to the membrane that is exposed to the repeated challenges and does not affect the responses to ATP in the contralateral side.
|
Receptors involved. In Fig. 4, we determined whether the responses to UTP and 2-MeSATP, relatively specific agonists of the P2u and P2y receptors, undergo the similar course of desensitization and recovery as in the ATP-stimulated responses. In addition, we determined whether the desensitization and recovery induced by each agonist can cross-affect the responses to the other. The ATP- and UTP-induced responses showed the same course of desensitization (Fig. 4, top left and middle). The pattern of the response to single UTP exposures during the initial, desensitized, and recovery phases of the response to repeated exposures to ATP mimicked those of ATP (Fig. 4, bottom left). Likewise, the pattern of the response to single ATP exposures during the initial, desensitized, and recovery phases of repeated UTP exposures mimicked those of UTP (Fig. 4, bottom middle). In contrast, the response to 2-MeSATP was small (usually <30% of the responses to the same concentration of ATP or UTP), failed to show the recovery process after desensitization, and did not affect the responses to ATP or UTP. These results indicate that the desensitization and recovery processes are mainly associated with the P2u or a P2u-like receptor-dependent mechanism.
|
Role of cAMP and adenylyl cyclase. The effect of the protein kinase A inhibitor H-89 (4) in Fig. 2 indicates that the production of cAMP and activation of protein kinase A plays a critical role in the stimulation of Isc by ATP. To elucidate further the role of cAMP-dependent mechanism in the desensitization and recovery of the ATP-stimulated Isc responses, we evaluated the effect of single doses of the membrane-permeable cAMP analog 8-BrcAMP during the initial, desensitized, and recovered phases induced by repeated doses of ATP. The magnitude of the response of Isc to 8-BrcAMP itself was relatively small (18.1 ± 3.1% of ATP-stimulated responses) and remained unchanged during the desensitization and recovery phases induced by repeated exposures to ATP. Importantly, the presence of 8-BrcAMP did not add to the ATP-stimulated responses during the initial or recovered phase (Fig. 5B). In sharp contrast, when added during the desensitized phase, 8-BrcAMP enhanced the ATP-stimulated Isc significantly (Fig. 5, A and B). These data lead us to hypothesize that the desensitization process of the Isc responses to repeated doses of ATP occurs via a second messenger system involving adenylyl cyclase-dependent cAMP production.
|
Changes in intracellular cAMP accumulation.
We determined intracellular cAMP accumulation to decipher directly the
involvement of adenylyl cyclase in the desensitization and recovery of
ATP stimulation of
Isc (Fig.
6). We measured intracellular cAMP content
in cells prepared in parallel with those used to record
Isc in the Ussing
chambers. Exposure to ATP for 3 min during the initial phase increased
intracellular cAMP content 4.1-fold. The stimulatory effect of ATP on
cAMP accumulation was attenuated to 1.6-fold in cells desensitized by
four repeated exposures to ATP (desensitized phase), but increased
again to 3.2-fold after recovery of the stimulation of
Isc by ATP
(recovered phase). This result indicates adenylyl cyclase activity
changes during the ATP-induced desensitization and recovery process.
Equipotent effects of the nonhydrolyzable ATP analog ATPS and
failure to stimulate cAMP accumulation by the
P1 receptor agonist adenosine
indicates that the P1 receptor is
not likely to be involved in the stimulation of cAMP production by ATP.
Addition of ATP into the basolateral bathing solution also stimulated
the production of cAMP, and it was not affected by the desensitization
and recovery process induced by the apically added ATP. This is
consistent with the result in the
Isc experiments
(Fig. 3) and confirms our hypothesis that the desensitization and
recovery process is limited to the one membrane that is exposed to the
repeated challenges. Figure 6 also shows that ATP-stimulated production
of cAMP is completely blocked in the presence of indomethacin, a
cyclooxygenase inhibitor, suggesting that the metabolic product of
arachidonic acid is involved importantly in the signaling mechanism to
stimulate adenylyl cyclase activity.
|
Effect of cholera toxin and pertussis toxin. To assess the involvement of G proteins in the cascade of events, we treated cells with cholera toxin or pertussis toxin (1 µg/ml for 3 h, apical side in the Ussing chambers) and observed the responses to repeated doses of ATP (Fig. 7A). The desensitization process was attenuated in cholera toxin-treated cells, supporting the idea that the decrease in adenylyl cyclase-dependent cAMP production is responsible for the desensitization of ATP-induced Isc responses. The desensitization process was also attenuated significantly in pertussis toxin-treated cells. We also evaluated the effect of pertussis toxin on the basal and ATP-stimulated cAMP production. Treatment of the cells with pertussis toxin itself did not affect the basal production of cAMP. It did, however, block the desensitization that occurs after repeated exposure to ATP (Fig. 7B). This suggests that a pertussis toxin-sensitive Gi protein-dependent mechanism might be involved.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is well known (29) and confirmed in our results that in MDCK cells
stimulation of
Isc by various
agonists including ATP occurs via an increase in net basolateral to
apical Cl secretion. In
addition, we have shown that ATP stimulates
Isc by mechanisms
involving both cAMP and intracellular
Ca2+ through the activation of
apical Cl
and basolateral
K+ conductances. The nature of the
Cl
secretory pathway is not
completely understood. CFTR or
Ca2+-activated
Cl
channels are not likely
to be involved primarily because neither glibenclamide nor DIDS affects
the ATP-stimulated
Isc response. Our
results are also consistent with the report showing that CFTR is not
expressed in these cells (31).
The desensitization of the ATP-stimulated Isc responses in our study was expected, but subsequent spontaneous recovery from desensitization with repeated challenges of ATP either in the apical or basolateral solution was an unexpected and interesting phenomenon. Our data showed that repeated challenges of ATP to a particular membrane side did not desensitize the response to contralateral application of ATP. The results indicate strongly that a membrane-specific mechanism, possibly the adenylyl cyclase system itself, is responsible for the desensitization and recovery process.
In a recent report (35), it was suggested that both the
P2u and
P2y receptors are involved in the
stimulation of
Isc by apically
added ATP in MDCK cells. In our study, the responses to ATP or UTP
displayed a similar course of desensitization and recovery and were
similarly cross-affected during the desensitization or recovery phase
induced by the other. In contrast, the specific P2y agonist 2-MeSATP evoked a
relatively small and transient effect that did not cross-affect the
responses to either ATP or UTP. These results suggest that the
P2u or a
P2u-like receptor is the major
transducer for the process of stimulation, desensitization, and the
recovery of Isc
induced by repeated exposure to ATP. The role of the adenylyl cyclase
system in the P2 purinergic
receptor-mediated response is still controversial. In this regard, some
contradictory results have been reported. Inhibition of cAMP
accumulation by activation of the
P2 purinergic receptor was
reported in rat hepatocytes (21), mouse ventricular myocytes (34),
FRTL-5 thyroid cells (26), and C6 glioma cells (19, 22). In contrast,
several reports suggested that the
P2-purinergic receptors could lead to increases in intracellular cAMP accumulation (12, 13, 17). Recently,
Post et al. (23) demonstrated the enhancement of cAMP production by the
P2 purinergic agonists in MDCK
cells. Our results also suggest the involvement of cAMP in the
signaling of ATP-induced stimulation of
Isc. Inhibition
by H-89 of both the initial and recovered responses of
Isc to ATP
suggests that a protein kinase A-dependent mechanism is involved in
both phases of stimulation. Determination of the changes in
intracellular cAMP production in response to various purinergic
agonists also supported our hypothesis. We can rule out possible
involvement of the P1 receptor in
ATP-stimulated cAMP production because the nonhydrolyzable ATP analog
ATPS as well as ATP stimulated
Isc, whereas the
P1 receptor agonist adenosine
failed to stimulate cAMP production. The action of ATP to stimulate
cAMP production may be mediated by cyclooxygenase-dependent metabolism
of arachidonic acid, because indomethacin inhibited almost completely
the ATP-stimulated
Isc and cAMP
production in both the initial and recovered phases. Prostaglandin
E2 is known to be a major product
of arachidonic acid metabolism in the kidney (3) and is a likely
candidate as the agonist in this stimulatory pathway.
We have provided several lines of evidence that the adenylyl cyclase
system is involved in the desensitization and recovery process of
ATP-stimulated
Isc. First,
addition of the membrane-permeable cAMP during the desensitized phase
enhanced the ATP-stimulated responses significantly, whereas it did not
during the initial or recovered phase. In addition, the desensitization
process was significantly attenuated in cholera toxin-treated cells.
These results suggest that the desensitization process is closely
linked with the decrease in intracellular cAMP concentration. The
absence of additive effect of the membrane-permeable cAMP on the
ATP-stimulated responses during the initial and recovered phases
implies that ATP can stimulate adenylyl cyclase sufficiently, resulting
in maximal activation of cAMP-dependent
Cl conductance in
nondesensitized cells. Second, the response to the membrane-permeable
cAMP remained unchanged during the ATP-induced desensitization and
recovery process. This indicates that
Cl
channels or another
mechanism that is distal to the production of cAMP is not affected
during the desensitization phase. This result, together with the
localization to the exposed membrane only of the desensitization
process of ATP-stimulated
Isc and cAMP
production, suggests strongly that a membrane-bound adenylyl cyclase is
affected during the desensitization process. Finally, determination of
ATP-stimulated cAMP production resulted in a similar course of
desensitization and recovery in parallel with the changes in the
responses of Isc.
This result confirms clearly our hypothesis that modification of the
adenylyl cyclase system is involved in the mechanism of the
desensitization and recovery of the ATP-induced stimulation of
Isc.
The molecular basis underlying the modification of the adenylyl cyclase activity during the ATP-induced desensitization and recovery is not clear. Attenuation of the desensitization process in pertussis toxin-treated cells suggests that a Gi protein might be involved in the desensitization mechanism. The effect of pertussis toxin is not likely to result from a nonspecific increase in intracellular cAMP, because it did not stimulate basal cAMP production by itself. So, it is suggested that the modification of Gi protein during the first several exposures to ATP attenuates the responsiveness of the adenylyl cyclase, which then recovers again with increasing number of repeated exposures.
In summary, our results suggest that the adenylyl cyclase system is
involved in the signaling mechanism of the ATP-stimulated of
Cl secretion and its
desensitization and recovery in MDCK cells. More detailed studies will
give us an insight into the understanding of purinergic
receptor-mediated responses and their signaling mechanism.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was funded by National Institutes of Health Grants DK-32753, DK-48977, and HL-47122 and the Cystic Fibrosis Foundation Research Development Program.
![]() |
FOOTNOTES |
---|
Address for reprint requests: W. B. Guggino, Professor of Physiology and Pediatrics, Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205.
Received 18 December 1996; accepted in final form 29 October 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, M. P.,
D. N. Sheppard,
H. A. Berger,
and
M. J. Welsh.
Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L1-L14,
1992
2.
Boarder, M. R.,
G. A. Weisman,
J. T. Turner,
and
G. F. Wilkinson.
G protein-coupled P2 purinoceptors: from molecular biology to functional responses.
Trends Pharmacol. Sci.
16:
133-139,
1995[Medline].
3.
Bonvalet, J.-P.,
P. Pradelles,
and
N. Farman.
Segmental synthesis and actions of prostaglandins along the nephron.
Am. J. Physiol.
253 (Renal Fluid Electrolyte Physiol. 22):
F377-F387,
1987
4.
Chijiwa, T.,
A. Mishima,
M. Hagiwara,
M. Sano,
K. Hayashi,
T. Inoue,
K. Naito,
T. Toshioka,
and
H. Hidaka.
Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells.
J. Biol. Chem.
265:
5267-5272,
1990
5.
Cliff, W. H.,
and
R. A. Frizzell.
Separate Cl conductances activated by cAMP and Ca2+ in Cl
-secreting epithelial cells.
Proc. Natl. Acad. Sci. USA
87:
4956-4960,
1990[Abstract].
6.
Connolly, T. M.,
W. J. Lawing, Jr.,
and
P. W. Majerus.
Protein kinase C phosphorylates human platelet inositol trisphosphate 5'-phosphomonoesterase, increasing the phosphatase activity.
Cell
46:
951-958,
1986[Medline].
7.
Dalziel, H. H.,
and
D. P. Westfall.
Receptors for adenine nucleotides and nucleosides: subclassification, distribution, and molecular characterization.
Pharmacol. Rev.
46:
449-466,
1994[Medline].
8.
Demolle, D.,
M. Lecomte,
and
J. M. Boeynaems.
Pattern of protein phosphorylation in aortic endothelial cells. Modulation by adenine nucleotides and bradykinin.
J. Biol. Chem.
263:
18459-18465,
1988
9.
Dho, S.,
K. Stewart,
and
J. K. Foskett.
Purinergic receptor activation of Cl secretion in T84 cells.
Am. J. Physiol.
262 (Cell Physiol. 31):
C67-C74,
1992
10.
Dubyak, G. R.,
and
C. El-Moatassim.
Signal transduction via P2-purinergic receptors for extracelluar ATP and other nucleotides.
Am. J. Physiol.
265 (Cell Physiol. 34):
C577-C606,
1993
11.
El-Moatassim, C.,
J. Dornand,
and
J.-C. Mani.
Extracellular ATP and cell signalling.
Biochim. Biophys. Acta
1134:
31-45,
1992[Medline].
12.
Gailly, P.,
B. Boland,
C. Paques,
B. Himpens,
R. Casteels,
and
J. M. Gillis.
Post-receptor pathway of the ATP-induced relaxation in smooth muscle of the mouse vas deferens.
Br. J. Pharmacol.
110:
326-330,
1993[Abstract].
13.
Griese, M.,
L. I. Gobran,
and
S. A. Rooney.
A2 and P2 purine receptor interactions and surfactant secretion in primary cultures of type II cells.
Am. J. Physiol.
261 (Lung Cell. Mol. Physiol. 5):
L140-L147,
1991
14.
Gstraunthaler, G.,
W. Pfaller,
and
P. Kotanko.
Biochemical characterization of renal epithelial cell cultures (LLC-PK1 and MDCK).
Am. J. Physiol.
248 (Renal Fluid Electrolyte Physiol. 17):
F536-F544,
1985
15.
Harden, T. K.,
J. L. Boyer,
and
R. A. Nicholas.
P2-purinergic receptors: subtype-associated signaling responses and structure.
Annu. Rev. Pharmacol. Toxicol.
35:
541-579,
1995[Medline].
16.
Harrison, S. M.,
and
D. M. Bers.
The effect of temperature and ionic strength on the apparent Ca-affinity of EGTA and the analogous Ca-chelators BAPTA and dibromo-BAPTA.
Biochim. Biophys. Acta
925:
133-143,
1987[Medline].
17.
Henning, R. H.,
M. Duin,
den A. Hertog,
and
A. Nelemans.
Characterization of P2-purinoceptor mediated cyclic AMP formation in mouse C2C12 myotubes.
Br. J. Pharmacol.
110:
133-138,
1993[Abstract].
18.
Hwang, T. H.,
E. M. Schwiebert,
and
W. B. Guggino.
Apical and basolateral ATP stimulate tracheal epithelial chloride secretion via multiple purinergic receptors.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1611-C1623,
1996
19.
Lin, W.-W.,
and
D. M. Chuang.
Endothelin- and ATP-induced inhibition of adenylyl cyclase activity in C6 glioma cells: role of Gi and calcium.
Mol. Pharmacol.
44:
158-165,
1993[Abstract].
20.
Mason, S. J.,
A. M. Paradiso,
and
R. C. Boucher.
Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium.
Br. J. Pharmacol.
103:
1649-1656,
1991[Abstract].
21.
Okajima, F.,
Y. Tokumitsu,
Y. Kondo,
and
M. Ui.
P2-purinergic receptors are coupled to two signal transduction systems leading to inhibition of cAMP generation and to production of inositol triphosphate in rat hepatocytes.
J. Biol. Chem.
262:
13483-13490,
1987
22.
Pianet, I.,
M. Merle,
and
J. Labouesse.
ADP and, indirectly, ATP are potent inhibitors of cAMP production in intact isoproterenol-stimulated C6 glioma cells.
Biochem. Biophys. Res. Commun.
163:
1150-1157,
1989[Medline].
23.
Post, S. R.,
J. P. Jacobson,
and
P. A. Insel.
P2 purinergic receptor agonists enhance cAMP production in Madin-Darby canine kidney epithelial cells via autocrine/paracrine mechanism.
J. Biol. Chem.
271:
2029-2032,
1996
24.
Raymond, J. R.
Multiple mechanisms of receptor-G protein signaling specificity.
Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol. 38):
F141-F158,
1995
25.
Ryu, S.-H.,
U.-H. Kim,
M. I. Wahl,
A. B. Brown,
G. Carpenter,
K.-P. Huang,
and
S. G. Rhee.
Feedback regulation of phospholipase C-beta by protein kinase C.
J. Biol. Chem.
265:
17941-17945,
1990
26.
Sato, K.,
F. Okajima,
and
Y. Kondo.
Extracellular ATP stimulates three different receptor-signal transduction systems in FRTL-5 thyroid cells.
Biochem. J.
283:
282-287,
1992.
27.
Sheppard, D. N.,
and
M. J. Welsh.
Inhibition of the cystic fibrosis transmembrane conductance regulator by ATP-sensitive K+ channel regulators.
Ann. NY Acad. Sci.
707:
275-284,
1993[Medline].
28.
Simmons, N. L.
Stimulation of Cl secretion by exogenous ATP in cultured MDCK epithelial monolayers.
Biochim. Biophys. Acta
646:
231-242,
1981[Medline].
29.
Simmons, N. L.
Renal epithelial Cl secretion.
Exp. Physiol.
78:
117-137,
1993[Medline].
30.
Strabel, D.,
and
M. Diener.
Evidence against direct activation of chloride secretion by carbachol in the rat distal colon.
Eur. J. Pharmacol.
274:
181-191,
1995[Medline].
31.
Stutts, M. J.,
C. M. Canessa,
J. C. Olsen,
M. Hamrick,
J. A. Cohn,
B. C. Rossier,
and
R. C. Boucher.
CFTR as a cAMP-dependent regulator of sodium channels.
Science
269:
847-850,
1995[Medline].
32.
Valentich, J. D.
Morphological similarities between the dog kidney cell line MDCK and the mammalian cortical collecting tubule.
Ann. NY Acad. Sci.
372:
394-405,
1981.
33.
Wilkinson, G. F.,
J. R. Purkiss,
and
M. R. Boarder.
Differential heterologous and homologous desensitization of two receptors for ATP (P2Y purinoceptors and nucleotide receptors) coexisting on endothelial cells.
Mol. Pharmacol.
45:
731-736,
1994[Abstract].
34.
Yamada, M.,
Y. Hamamori,
H. Akita,
and
M. Yokoyama.
P2-purinoceptor activation stimulates phosphoinositide hydrolysis and inhibits accumulation of cAMP in cultured ventricular myocytes.
Circ. Res.
70:
477-485,
1992[Abstract].
35.
Zegra-Moran, O.,
G. Romeo,
and
L. J. V. Galietta.
Regulation of transepithelial ion transport by two different purinoceptors in the apical membrane of canine kidney (MDCK) cells.
Br. J. Pharmacol.
114:
1052-1056,
1995[Abstract].