Laboratory of Physiology, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422 - 8526, Japan
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ABSTRACT |
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Extracellular ATP regulates a variety of functions in epithelial tissues by activating the membrane P2-receptor. The purpose of this study was to investigate the autocrine/paracrine regulation by luminal ATP of electrogenic amiloride-sensitive Na+ absorption in the distal colon from guinea pigs treated with aldosterone by measuring the amiloride-sensitive short-circuit current (Isc) and 22Na+ flux in vitro with the Ussing chamber technique. ATP added to the luminal side inhibited the amiloride-sensitive Isc and 22Na+ absorption to a similar degree. The concentration dependence of the inhibitory effect of ATP on amiloride-sensitive Isc had an IC50 value of 20-30 µM, with the maximum inhibition being ~50%. The effects of different nucleotides and of a nucleoside were also studied, the order of potency being ATP = UTP > ADP > adenosine. The effects of ATP were slightly, but significantly, reduced in the presence of suramin in the luminal solution. The inhibitory effect of luminal ATP was more potent in the absence of both Mg2+ and Ca2+ from the luminal solution. Pretreatment of the tissue with ionomycin or thapsigargin in the absence of serosal Ca2+ did not affect the percent inhibition of amiloride-sensitive Isc induced by ATP. Mechanical perturbation with a hypotonic luminal solution caused a reduction in amiloride-sensitive Isc, this effect being prevented by the presence of hexokinase, an ATP-scavenging enzyme. These results suggest that ATP released into the luminal side by hypotonic stimulation could exert an inhibitory effect on the electrogenic Na+ absorption. This effect was probably mediated by a P2Y2 receptor on the apical membrane of colonic epithelial cells, and a change in the intracellular Ca2+ concentration may not be necessary for this process.
P2Y receptor; UTP; volume regulation; intestinal absorption; intracellular Ca2+
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INTRODUCTION |
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ATP, WHICH IS RELEASED FROM many cell types including epithelial tissue, functions as an autocrine/paracrine agonist by activating the two large families of P2X and P2Y receptors (41). The P2Y receptors belong to the superfamily of G protein-coupled receptors and are coupled to the inositol phosphate pathway resulting in the mobilization of Ca2+. A subset of P2Y receptors is activated by UTP to a similar degree to that by ATP and, in some instances, more potently (28, 41, 52).
The colon, the terminal part of the gastrointestinal tract, performs
both the absorption and secretion of a variety of electrolytes by the
epithelial transport systems. These transport systems are regulated by
many kinds of endocrine, neurocrine, and paracrine agents and probably
play an important role in maintaining fluid and electrolyte homeostasis
in the whole body (1). Previous studies have suggested
that P2-type receptor activation by ATP (and/or UTP) was also involved
in regulating electrolyte transport in the colon. It has been reported
that ATP indirectly stimulated colonic Cl secretion by
acting on submucosal secretomotor neurons (9). More
recently, ATP has been shown to induce an increase in intracellular Ca2+ concentration ([Ca2+]i) and
to stimulate Cl
and mucin secretion in a colonic
carcinoma cell line (12, 17, 18, 21, 23, 32, 37, 46, 47).
In the cases of T84 and Caco-2 cells, UTP added to the luminal side
also elicited anion secretion, implicating the role of the P2Y-type
receptor residing on the luminal membrane (23, 46). In
addition to anion secretion, the induction of electrogenic
K+ secretion by luminal ATP and UTP through activation of
the P2Y receptor has been reported in the rat colon (26).
The epithelial P2Y receptor in the colon may not only participate in
regulating transport functions, but may also play a role in regulating
cell proliferation and apoptosis (21).
Electrogenic Na+ absorption, which involves an apical
amiloride-sensitive Na+ channel and basolateral
Na+, K+-ATPase/K+ channel, is one
of the major pathways for Na+ absorption in the colon
(1). Although this Na+ transport pathway is
known to be stimulated by aldosterone and -adrenergic agonists and
inhibited by vasopressin (1, 44, 51), the role of
extracellular ATP and other nucleotides in regulating this process has
not been reported. ATP and/or UTP, however, has been shown to inhibit
electrogenic Na+ absorption from the luminal side in
several other epithelia by activating, in most cases, P2Y receptors
(3, 8, 10, 22, 24, 27, 34, 36, 42, 53). We, therefore,
investigated whether a functional P2Y receptor exists on the apical
membrane of the colonic epithelium, which would be coupled to the
regulation on electrogenic Na+ absorption. To this end, the
effect of ATP and UTP added to the luminal side on the short-circuit
current (Isc) and 22Na+
flux was examined in an isolated guinea pig distal colon mounted in
Ussing chambers. In addition, we examined the effect of applying a
hypotonic luminal solution to clarify whether endogenous ATP may
support the regulatory volume decrease by inhibiting Na+ uptake.
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MATERIALS AND METHODS |
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Tissue preparation. Hartley-strain, male guinea pigs weighing 250-700 g were used in the experiments. All animals were injected twice with 375 µg/kg body wt of aldosterone (1.39 mM in saline) to enhance the electrogenic sodium absorption, once by a subcutaneous injection the evening before the experiment, and then by an intramuscular injection 4 h before the start of the experiment. The animals were stunned by a blow to the head and bled to death. The distal colon was excised, opened along the root of the mesentery, and muscle and submucosal layers were removed with a glass microscopic slide (55). Procedures involving animals were approved by the Institutional Animal Care Board at the University of Shizuoka.
Solutions. The standard bathing solution contained (in mM) 119 NaCl, 21 NaHCO3, 2.4 K2HPO4, 0.6 KH2PO4, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose. The Ca2+-free solution was prepared by omitting CaCl2 and adding 0.2 mM EGTA. The Ca2+-Mg2+-free solution omitted both CaCl2 and MgCl2, whereas 1 mM EDTA/5 mM Tris was added. The isotonic control solution for the mechanical stimulation experiment contained (in mM) 88 NaCl, 21 NaHCO3, 2.4 K2HPO4, 0.6 KH2PO4, 1.2 MgCl2, 1.2 CaCl2, 10 glucose, and 60 sucrose (290 mosmol/KgH2O). The hypotonic solution was prepared by omitting sucrose from the isotonic solution (230 mosmol/KgH2O). All solutions were gassed with 95% O2-5% CO2 (pH 7.3-7.4).
Isc measurements. Isc and transmural tissue resistance (Gt) were measured in vitro in Ussing chambers as previously described (44, 55). Stripped mucosa was mounted vertically between acrylic resin chambers with an internal surface area of 0.5 cm2. The bathing solution in each chamber was 10 ml, and its temperature was maintained at 37°C in a water-jacketed reservoir. The tissue was continuously short circuited, with compensation for the fluid resistance between the two potential-sensing bridges, by using a voltage-clamping amplifier (CEZ9100, Nihon Kohden, Tokyo, Japan). The transepithelial potential was measured through 1 M KCl-agar bridges connected to a pair of calomel half-cells, the transepithelial current being applied across the tissue through a pair of Ag-AgCl electrodes kept in contact with the mucosal and serosal bathing solutions through a pair of 1 M NaCl-agar bridges. The Isc value is expressed as positive when the current flowed from the mucosa to serosa. Gt was measured by recording the current resulting from short-duration, square bipolar voltage pulses (±5 mV) imposed across the tissue and then calculated according to Ohm's law. In all experiments, 10 µM indomethacin was added to the serosal solution to prevent the effect of endogenously produced prostaglandins.
22Na+ flux
measurements.
The unidirectional transmural flux of 22Na+ was
measured under short-circuit conditions. The mucosal-to-serosal
(Jms) and serosal-to-mucosal (Jsm) flux values were measured in adjacent
tissues that had Gt values differing by <30%.
Thirty minutes were allowed for the isotopic steady state to be reached
after labeling either the serosal or mucosal side of the bathing
solution with 22Na+. Ten samples (0.5 ml each)
were taken from the unlabeled side at 10-min intervals and replaced
with an equal volume of the unlabeled solution. The medium samples were
assayed for 22Na+ by a -well spectrometer.
Materials. Indomethacin, amiloride, benzamil, bumetanide, ATP, UTP (Na+ salt), ADP (Na+ salt), adenosine, suramin, thapsigargin, and hexokinase were purchased from Sigma (St. Louis, MO). ATP-Na+ was used in the experiment to examine the effect of luminal Ca2+-Mg2+ removal, with ATP-Mg2+ being used in all other experiments. Ionomycin was purchased from Calbiochem (San Diego, CA). All other chemicals were purchased from Wako Pure Chemicals (Osaka, Japan). Each drug was applied from a concentrated stock solution dissolved in water or DMSO, the final volume of DMSO in an experimental solution always being 0.1%. 22Na+ was purchased from Dupont New England Nuclear (Boston, MA).
Statistics analyses. Each data value is presented as the mean ± SE of number (n) of guinea pigs. Statistical comparisons between two means were made with Student's t-test (paired or unpaired, as appropriate). Significance was accepted at P < 0.05.
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RESULTS |
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Effect of luminal ATP on the electrogenic
Na+ absorption.
The addition of ATP (1 mM) to the mucosal solution resulted in a
decrease in Isc and Gt in
the guinea pig distal colon (Fig. 1,
A and C). In some cases, a small transient
Isc increase with a duration of <5 min could be
observed before the decrease. After the tissue had been pretreated with
the epithelial Na+ channel blocker amiloride (0.1 mM
mucosal), no such ATP-induced Isc and
Gt decreases were apparent, although initial
transient Isc increase was enhanced (Fig. 1,
B and D). These results suggest that the luminal
ATP-induced reduction in Isc and
Gt was probably due to an inhibition of the
amiloride-sensitive electrogenic Na+ absorption. On the
other hand, when ATP was added to the serosal side (1 mM), there was a
robust increase in Isc and
Gt without any Isc
decrease below the basal level (Fig. 2;
Isc = 110 ± 11 µA/cm2,
Gt = 2 ± 0.4 mS/cm2,
n = 4). Therefore, ATP added to the mucosal solution
probably caused the decrease in Isc through the
activation of a receptor on the apical surface, and not a receptor on
the basolateral surface, after penetrating into the serosal solution.
Negative Isc in the presence of amiloride (Fig.
1) is likely to be mainly due to an electrogenic K+
secretion, because it was largely suppressed by serosal bumetanide (see
Figs. 5-7 and also Ref. 44). Whether the
K+ secretion was also affected by luminal ATP remained to
be determined.
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Agonists and antagonists.
The potency of different nucleosides or nucleotides for reducing
amiloride-sensitive Isc was then studied. This
and the subsequent experiments were conducted in the presence of
serosal bumetanide (10 µM) to inhibit the Isc
components due to electrogenic K+ and Cl
secretions. Under this condition, basal Isc is
almost totally amiloride-sensitive (44). As is shown in
Fig. 4, the concentration-response relationship for the inhibitory effect of luminal ATP on
amiloride-sensitive Isc in the presence of
bumetanide was essentially similar to that observed in the absence of
serosal bumetanide (Fig. 3). Luminal UTP inhibited amiloride-sensitive
Isc with a similar potency to that of ATP,
suggesting the involvement of a P2Y receptor (28, 41, 52).
The threshold concentration of ADP was ~10-fold higher and that of
adenosine was ~100-fold higher than the threshold concentration of
ATP or UTP. Thus the order of potency appears to have been ATP = UTP > ADP > adenosine.
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Role of [Ca2+]i.
The activation of P2Y receptors is, in most cases, coupled to the
Ca2+-signaling pathway. The next series of experiments was
designed to investigate the role of the increase in
[Ca2+]i in the ATP-induced inhibition of
Na+ absorption. We first examined whether or not an
increase in [Ca2+]i by itself could inhibit
amiloride-sensitive Isc in the guinea pig colon
by using the Ca2+ pump inhibitor thapsigargin and the
Ca2+ ionophore ionomycin. Both thapsigargin and ionomycin
caused a decrease in Isc (Fig.
6). When the tissue had been pretreated with luminal amiloride (0.1 mM), neither thapsigargin nor ionomycin had
any effect on Isc (data not shown). In addition,
the Isc decreases induced by thapsigargin and
ionomycin were remarkably attenuated when the tissue was bathed with a
serosal Ca2+-free solution (see Fig.
7, C and D). These
findings indicate that an increase in [Ca2+]i
was sufficient to cause a decrease in the amiloride-sensitive Na+ absorption.
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ATP release to the luminal solution.
In the final series of experiments, we investigated the physiological
source of ATP acting on the apical membrane receptor coupled with the
inhibition of electrogenic Na+ absorption in the colon. We
first examined the effect on basal Isc of adding
hexokinase to the luminal solution. If sufficient ATP is released to
the luminal solution to induce inhibition under the basal condition,
the addition of hexokinase would evoke an increase in
Isc by scavenging ATP as a result of
respectively converting ATP and the glucose to ADP and
glucose-6-phosphate (30). However, as shown in Fig.
8A, hexokinase (2.5 U) added to the luminal solution failed to increase Isc,
and instead slightly decreased it (by 4.5 ± 1.2%,
n = 10). In the presence of hexokinase at this
concentration, the inhibition of Isc induced by
luminal 10 µM ATP was completely abolished (data not shown,
n = 6), and that induced by 100 µM ATP was greatly
suppressed (Fig. 8A; 4.8 ± 2.1% inhibition,
n = 3; compare with Fig. 4). It is thus unlikely under
these experimental conditions that sufficient ATP to inhibit electrogenic Na+ absorption would have been released to the
luminal solution.
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DISCUSSION |
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The results of our present study on the guinea pig distal colon
treated with aldosterone suggest that a P2Y-type purinergic receptor is
likely to be present on the apical membrane that is coupled to the
inhibition of electrogenic Na+ absorption. ATP added to the
mucosal side caused decreases in Isc (and
Gt) and 22Na+ absorption
of essentially similar magnitude, and the Isc
and Gt decreases induced by ATP were suppressed
in the presence of amiloride. UTP, which is an agonist for a subset of
P2Y receptor, also partially inhibited amiloride-sensitive
Isc. In the present preparation, luminal ATP
caused, in addition to the ultimate decrease, a transient increase in
Isc, which was not inhibited by amiloride (Fig.
1), suggesting the stimulation of Cl secretion. Whether
this response would also be mediated by a P2Y receptor remains to be
determined. In addition, the present preparations exhibited
electrogenic K+ secretion (Fig. 1). Whether this process is
also affected by luminal ATP remains to be examined. Previous studies
on gastrointestinal tissues have suggested that stimulation of the
apical P2Y receptor induced Cl
and K+
secretion (7, 23, 26, 46).
Multiple subtypes of P2Y receptors have been found (28, 41,
52), and it has been suggested that the P2Y-receptor subtype in
the apical membrane is P2Y2 for colonic K+
secretion (26) and P2Y4 for intestinal
Cl secretion (7, 31). To determine the
subtype of the apical membrane P2Y receptor responsible for inhibiting
Na+ absorption in the colon, the potency order of different
nucleotides or nucleosides for suppressing amiloride-sensitive
Isc was examined, the result being ATP = UTP > ADP > adenosine. In addition, suramin significantly
attenuated the inhibitory effect of luminal ATP on amiloride-sensitive
Isc. These findings are at least consistent with
the P2Y2 receptor contributing to the inhibition of
Na+ absorption (5, 14, 33, 41, 52). However,
it has recently been reported that the P2Y4 receptor in the
rat and mouse, but not human, was activated equipotently by ATP and UTP
(2, 31, 48). In addition, suramin, although only weakly,
inhibited the rat, but not human or mouse, P2Y4 receptor
(2, 6, 48). Thus the possible involvement of the
P2Y4 receptor cannot be entirely excluded. It has been said
that the agonist potency determined from a functional assay on the
native or recombinant P2 receptor was difficult to interpret for
several reasons (29, 52). For example, it may be necessary
to determine the effect of an ectonucleotidase, possibly existing on
the apical surface of the colon (see the next paragraph for further
discussion). In addition, more than one subtype of P2 receptor might be
present on the apical membrane and contribute to the ATP-induced
inhibition of Na+ absorption (see final paragraph). Further
studies are therefore clearly needed to determine the receptor subtype
responsible for the luminal ATP-induced inhibition of Na+ absorption.
When both Ca2+ and Mg2+ were removed from
the mucosal side, the potency of the inhibitory effect of ATP on
amiloride-sensitive Isc was enhanced (Fig. 3).
One possible explanation for this is that the active form of the
agonist that induced the inhibition of Na+ absorption was
ATP4 and not MgATP2
or CaATP2
(56). However, it is more likely that this enhanced effect by Ca2+ and Mg2+ removal was due to the
inhibition of ectonucleotidases on the apical surface. The activities
of certain ectonucleotidases depend on the presence of Ca2+
or Mg2+ or both (19, 40). The activity of such
ectonucleotidases that degrade nucleotides could skew the
concentration-response curve by generally shifting it to the right. In
fact, the potency of the inhibition of Na+ absorption by
luminal ATP in the present study was shifted to the right by at least
one order of magnitude compared with the potency on the cloned P2Y
receptors in the expression system (2, 14, 33). Although a
previous study has demonstrated that cultured colonic epithelial cells
possessed ecto-ATPase activities (12, 16, 47), whether or
not such an ectonucleotidase was present on the apical surface of the
present preparation remains to be determined.
The activation of P2Y receptors is known to lead to an increase in [Ca2+]i through activation of the phospholipase C inositol 1,4,5-trisphosphate pathway (41, 52). In addition, in the present colon preparation, the amiloride-sensitive Na+ absorption could be inhibited by an increase in [Ca2+]i, because it was inhibited by the addition of ionomycin or thapsigargin (Fig. 6). It seems, therefore, reasonable to assume that an increase in [Ca2+]i mediated the ATP-induced inhibition of Na+ absorption. However, the removal of Ca2+ from the serosal side or the addition of thapsigargin or ionomycin in the absence of serosal Ca2+ failed to affect the ATP-induced inhibition of amiloride-sensitive Isc (Fig. 7). Under these conditions, a receptor activation-coupled [Ca2+]i increase was presumably attenuated by depletion of the [Ca2+]i store (4, 39, 49). Therefore, an intracellular signaling pathway other than (or in addition to) the [Ca2+]i increase was activated and may mainly have been responsible for the ATP-induced inhibition of electrogenic Na+ absorption. It has previously been shown in several tissues that an elevated [Ca2+]i level was not required for the responses induced by activation of the P2Y receptor (15, 17, 22, 25, 27, 35).
Extracellular ATP released by mechanical or hypotonic stress has been
suggested to have importance in the autocrine/paracrine control of
epithelial cell functions (11, 13, 16, 20, 38, 43, 45, 50,
54). The present results for the colon suggest that a hypotonic
challenge on the mucosal side caused a release of ATP, which, in turn,
inhibited amiloride-sensitive Na+ absorption, probably via
interaction with the apical P2Y receptor (Fig. 8). The inhibition of
Na+ entry would conceivably lead to a reduction in the
epithelial cell volume, thereby facilitating a regulatory volume
decrease of the cell after the swelling induced by the hypotonic
challenge. Facilitation of the regulatory volume decrease mediated by a
released nucleotide has been demonstrated in gastrointestinal and
airway epithelial cells (11, 43, 54). In addition, the
inhibition of Na+ absorption in concert with the
stimulation of Cl secretion, which is probably also
induced by luminal ATP (Fig. 1), might help in flushing out noxious
agents from the colonic lumen. In many other epithelial cells, luminal
ATP has been reported to inhibit electrogenic Na+
absorption and to stimulate electrogenic Cl
secretion
through acting on the apical P2Y receptor (8, 10, 22, 24, 36, 42,
53). It is now clear in several epithelial cells and is likely
to also be the case in the colon that, after mechanical/hypotonic
stress, nucleotides are released to both the mucosal and serosal sides
(28, 43, 50). In addition, multiple subtypes of the
purinergic receptors are generally expressed on both apical basolateral
membranes (7, 12, 23, 27, 36, 46). Autocrine/paracrine
regulation of the intestinal transport function by nucleotides is thus
a very complex issue and warrants further study.
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ACKNOWLEDGEMENTS |
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We thank S. Ueno for expert technical assistance and T. Innes for help in editing the English text.
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FOOTNOTES |
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This work was supported, in part, by Salt Science Research Foundation Grants 9435 and 9537 and, in part, by a Sasakawa scientific research grant from The Japan Science Society.
Address for reprint requests and other correspondence: Y. Suzuki, Laboratory of Physiology, School of Food and Nutritional Sciences, Univ. of Shizuoka, Shizuoka 422-8526, Japan (E-mail: yuichi{at}smail.u-shizuoka-ken.ac.jp).
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.
March 6, 2002;10.1152/ajpgi.00541.2001
Received 26 December 2001; accepted in final form 8 February 2002.
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