No evidence for inhibition of ENaC through CFTR-mediated release of ATP

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DOI: 10.1016/S0005-2736(02)00502-3 · Source: PubMed
Abstract
Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl(-) secretion and inhibit amiloride-sensitive Na(+) transport. CFTR has been suggested to conduct adenosine 5'-triphosphate (ATP) or to control ATP release to the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na(+) channels (ENaC) could be by release of ATP or uridine 5'-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after coexpression of P2Y(2) receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl(-) channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl(-) transport or inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N(2),2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate (cGMP) and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl(-).
No evidence for inhibition of ENaC through
CFTR-mediated release of ATP
Jens Ko¨nig
a
, R. Schreiber
a
, M. Mall
b
, K. Kunzelmann
a,
*
a
Department of Physiology and Pharmacology, University of Queensland, St. Lucia, Brisbane, QLD 4072, Australia
b
Cystic Fibrosis/Pulmonary Research and Treatment Center, School of Medicine, The University of North Carolina at Chapel Hill,
7011 Thurston Bowles Building, Chapel Hill, NC 27599-7248, USA
Received 27 February 2002; received in revised form 4 June 2002; accepted 26 June 2002
Abstract
Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl
secretion and
inhibit amiloride-sensitive Na
+
transport. CFTR has been suggested to conduct adenosine 5V-triphosphate (ATP) or to control ATP release to
the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na
+
channels (ENaC)
could be by release of ATP or uridine 5V-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this
question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and
extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after
coexpression of P2Y
2
receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl
channel blocker 4,4V-
diisothiocyanatostilbene-2,2V-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl
transport or
inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N
2
,2V-O-dibutyrylguanosine 3V,5V-cyclic monophosphate (cGMP)
and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the
inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl
.
D 2002 Elsevier Science B.V. All rights reserved.
Keywords: CFTR; ENaC; Xenopus oocytes; Trachea; Colon; Cystic fibrosis; ATP; UTP; Purinergic receptor; Epithelial transport
1. Introduction
Amiloride-sensitive epithelial Na
+
channels (ENaC) are
essential for electrogenic absorption of Na
+
in kidney,
airways, intestine and other epithelial tissues [6]. ENaC is
expressed in airway and colonic epithelial cells, together
with cystic fibrosis transmembrane conductance regulator
(CFTR) Cl
channels [18]. CFTR is not only a Cl
channel but it also controls the activity of numerous other
ion transport proteins [15,35]. It has been demonstrated that
ENaC is inhibited by CFTR in airway and coloni c epithelial
tissues and cells coexpressing both proteins recombinantly
[19,21]. Others have been unable to demonstrate inhibition
of ENaC by CFTR in Xenopus oocytes or demonstrated
activation of ENaC by CFTR in the sweat duct epithelium.
These differences are likely due to variable relative expres-
sion levels of CFTR and ENaC in Xenopus oocytes and
indicate functional differences between the different epithe-
lial tissues, respectively [14]. We have postulated that
activation of CFTR Cl
channels and simultaneous inhib-
ition of ENaC results in a switch of epithelial ion transport
from absorption under control conditions, towards secretion
after stimulation by secretagogues [15,18].
In a surprising similarity to the regulation of ENaC by
CFTR, an inverse regulation of Cl
and Na
+
transport has
been found for the stimulation of purinergic P2Y receptors,
which are colocalized in respiratory epithelial cells together
with ENaC [2,27,32]. Binding of adenosine 5V-triphos-
phate (ATP) or uridine 5V-triphosphate (UTP) to P2Y
receptors leads to an increase in intracellular Ca
2+
and
0005-2736/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S 0005-2736(02)00502-3
Abbreviations: CFTR, cystic fibrosis transmembrane conductance
regulator; IBMX, 3-isobutyl-1-methylxanthine; cAMP, 5Vcyclic adenosine
monophosphate; ENaC, epithelial Na
+
channel; UTP, uridine 5V-triphos-
phate; ATP, adenosine 5V-triphosphate; PKG, protein kinase G; cGMP,
N
2
,2V-O-dibutyrylgu anosin e 3V,5V-cyclic monop hosp hate; DIDS, 4,4V-
diisothiocyanatostilbene-2,2V-disulfonic acid; PKG (peptide) inhibitor, H-
Arg-Lys-Arg-Ala-Arg-Lys-glu-OH
*
Corresponding author. Tel.: +61-7-3365-4104; fax: +61-7-3365-1766.
E-mail addresses: mmall@med.unc.edu (M. Mall),
uqkkunze@mailbox.uq.edu.au (K. Kunzelmann).
www.bba-direct.com
Biochimica et Biophysica Acta 1565 (2002) 17 28
activation of Ca
2+
-dependent Cl
channels (CaCC) [27].
In parallel, purinergic agonists attenuate amiloride-sensitive
Na
+
transport, probably by inhibition of ENaCs [2,27,32].
For both CFTR and purinergic inhi bition of ENaC, the
underlying mechanism s have not yet been described in
detail [17]. Some requirements have been identified, such
as a functional first nucleotide binding domain (CFTR) or
G protein function (P2Y receptor) [16,20]. Along this line,
Cl
transport induced by CFTR Cl
channels or P2Y
2
-
activated CaCC channels plays a crucial role [14,20].
These similarities suggest a common link for CFTR and
purinergic inhibition of ENaC. Such a link could exist
through the control of ATP transport by CFTR [11,36,37].
ATP transport by CFTR, however, is a controversial issue.
It has been detected in some but not in other studies
[7,8,22,23,33,38,43]. ATP transport by CFTR or control of
an ATP transporting protein by CFTR [1] should lead to 5V
cyclic adenosi ne monophosphate (cAMP)-mediated release
of ATP or UTP, stimulation of Ca
2+
-activated Cl
channels
and inhibition of ENaC. We examined this possibility and
other ATP-dependent regulatory pathways in the present
study. We found no evidence for a CFTR-mediated release
of purinergic agonists and a functional link of both puriner-
gic and CFTR-dependent inhibition of ENaC via the pro-
posed mechanism.
2. Methods
2.1. cRNAs for ENaC, CFTR, P2Y
2
and NDPK and
expression in Xenopus oocytes
cDNA encoding rat (a,h,g) ENaC (kindly provided by
Prof. Dr. B. Rossier, Pharmacological Institute of Lau-
Fig. 1. Expression of ENaC and CFTR in Xenopus oocytes. (A) Continuous recording of amiloride-sensitive whole cell conductance ( G
Amiloride
) over 90 min
does not show any run down of G
Amiloride
. (B) Original recordings of the whole cell currents obtained in a CFTR/ENaC expressing oocyte. Activation of CFTR
by stimulation with IBMX (1 mmol/l) and forskolin (2 Amol/l) inhibits the amiloride-sensitive whole cell current. The inhibition shows a partial recovery of
G
Amiloride
15 min after omission of IBMX/forskolin. (C) G
Amiloride
can be inhibited repetitively by activating and deactivating CFTR. * indicate significant
difference from control (paired t-test). (Number of experiments).
J. Ko
¨
nig et al. / Biochimica et Biophysica Acta 1565 (2002) 17–2818
sanne, Switzerland), human CFTR, mouse nucleoside
diphosphate kinase A (NDPK) and rat P2 Y
2
receptor
were linearized in pBluescript or pTLN [12] with NotI
or MluI, and in vitro transcribed using T7, T3 or SP6
promotor and polymerase (Message Machine, Ambion,
USA). Isolation and microinjection of oocytes have been
described in a previous report [9]. In brief, after isolatio n
from adult Xenopus laevis female frogs (Xenopus express,
South Africa), oocytes were dispersed and defolliculated
by a 0.8-h treatment with collagenase (type A, Boeh-
ringer, Germany). Subsequently, oocytes were rinsed and
kept at 18 jC in ND96-buffer (in mmol/l): NaCl 96, KCl
2, CaCl
2
1.8, MgCl
2
1, HEPES 5, Na-pyruvate 2.5, pH
7.55), supplemented with theophylline (0.5 mmol/l) and
gentamycin (5 mg/l).
2.2. Double electrode voltage clamp
Oocytes were injected with cRNA (110 ng) after
dissolving in about 50 nl double-distilled water (Nanoliter
Injector WPI, Germany). Water injected oocytes served as
controls. Two to four days after injection, oocytes were
impaled with two electrodes (Clark Instruments), which had
a resistance of < 1 MV when filled with 2.7 mol/l KCl. Two
bath electrodes were used, which had resistances of 1.7 and
2.2 kV, respectively, when immersed in ND96 bath sol-
ution. Using two bath electrodes and a virtual-ground head-
stage, the voltage drop across R
serial
was effectively zero.
Membrane currents were measured by voltage clamping of
the oocytes (Warner oocyte clamp amplifier OC725C) in
intervals from 90 to + 30 mV, in steps of 10 mV, each 1 s.
Fig. 2. Effects of stimulation of luminal purinergic receptors by ATP or UTP on ion transport in mouse trachea and Xenopus oocytes expressing ENaC. (A)
Continuous recording of the transepithelial voltage (V
te
) in mouse trachea. ATP (100 Amol/l) induced a transient voltage deflection and inhibited amiloride (A)
sensitive transport. (B) Summary of the amiloride-sensitive short circuit currents before and after stimulation with ATP. (C) Whole cell currents measured in
Xenopus oocytes expressing the epithelial Na
+
channel ENaC and effects of amiloride (10 Amol/l) in the absence or presence of ATP (100 Amol/l). (D)
Summary of the amiloride-sensitive whole cell conductances ( G
Amil
) measured in ENaC expressing oocytes. Stimulation by ATP or UTP (both 100 AM) has no
impact on G
Amil
. (Number of experiments).
J. Ko
¨
nig et al. / Biochimica et Biophysica Acta 1565 (2002) 17–28 19
Current data were filtered at 50 Hz. Data were collected
continuously (PowerLab, AD-Instruments, Australia) and
were analyzed by using the programs chart and scope
(PowerLab, AD-Instruments). Conductances were calcu-
lated according to Ohm’s law and amiloride-sensitive con-
ductances ( G
Amil
) is used in the present report to express the
amount of whole cell conductance that is inhibited by 10
Amol/l amiloride. During the whole experiment, the bath
was continuously perfused at a rate of 510 ml/min. All
experiments were conducted at room temperature (22 jC).
2.3. Ussing chamber experiments
Tracheas were taken from mice (Quackenbush, animal
facility of the University of Queensland) after sacrificing the
animal by cervical dislocation, and opened by a longitudinal
cut after connecti ve tissues were removed. Mouse distal
colon was removed from the animal and the mucosa was
separated mechanically from the submucosal tissue. Small
superfic ial m uc osal biopsies were obt aine d fr om human
rectum of 11 individuals (Department of Pediatrics, Univer-
sity of Freiburg, Germany). The study has been approved by
the ethical committee of the University of Freiburg, Germany.
Tissues were put immediately into a cold buffer solution of
the following composition (mmol/l): NaCl 145, KCl 3.8,
D-
glucose 5, MgCl
2
1, HEPES 5, Ca-gluconate 1.3. The tissues
were mounted into a modified Ussing chamber with a circular
aperture of 0.95 mm
2
. The luminal and basolateral sides of the
epithelium were perfused continuously at a rate of 10 ml/min
(chamber volume 2 ml). The bath solut ion had the following
composition (mmol/l): NaCl 145, KH
2
PO
4
0.4, K
2
HPO
4
1.6,
D-glucose 5, MgCl
2
1, HEPES 5, Ca-gluconate 1.3. pH was
adjusted to 7.4. Bath solutions were heated to 37 jC using a
water jacket. Experim ents were carried out under open circuit
conditions. Values for transepith elial voltages (V
te
)were
referred to the serosal side of the epithelium. Transepithelial
resistance (R
te
) was determined by applying short (1 s)
current pulses (DI = 0.5 AA). Voltage deflections obtained
Fig. 3. Effects of stimulation by IBMX (100 Amol/l)/forskolin (2 Amol/l) (I/F) and ATP or UTP (both 100 Amol/l) on transepithelial voltages and summary of
short circuit currents measured in mouse trachea. (A) Incubation with hexokinase (5 U/ml) and glucose (15 mM) attenuates ATP-activated transport but has no
effect on I/F induced secretion. (B) The P2Y blocker suramin (100 Amol/l) inhibits UTP-activated transport but has no effect on I/F-induced secretion. (C) The
Cl
channel blocker DIDS inhibits ATP-activated transport but has no effect on I/F-induced secretion. * indicate significant difference from control (paired t-
test). (Number of experiments).
J. Ko
¨
nig et al. / Biochimica et Biophysica Acta 1565 (2002) 17–2820
under conditions without the mucosa present in the chamb er
were subtracted from those obtained in the presence of the
tissues. R
te
was calculated according to Ohms law (R
te
= DV
te
/
DI). The equivalent short circuit current (I
sc
) was calculated
(I
sc
= V
te
/R
te
) and the amiloride-sensitive I
sc
(I
sc-Amil
) is used
to express the amount of equivalent short circuit current that
is inhibited by 10 Amol/l amiloride. Tissue preparations were
only accepted if the transepithelial resistance exceeded that
obtained for an empty chamber at least by a factor of 3.
Recordings were usually stable for 3 4 h.
2.4. Mate rials and statistical analysis
All used compounds were of highest available grade of
purity. ATP, UTP, suramin, carbachol, 3-isobutyl-1-methyl-
xanthine (IBMX), membrane-permeable N
2
,2V-O-dibutyryl-
Fig. 4. Summary of whole cell conductances measured in Xenopus oocytes coexpressing ENaC and P2Y
2
receptors. A whole cell Cl
conductance is activated
by stimulation with ATP (100 Amol/l) (A), which does not affect amiloride-sensitive Na
+
conductance ( G
Amil
) (B). (C E) Summary of whole cell
conductances measured in oocytes coexpressing ENaC, P2Y
2
receptors and CFTR. A whole cell Cl
conductance is activated by stimulation with ATP (100
Amol/l). Suramin itself has no effects on basal ion conductance (C) or amiloride sensitive conductance (D). However, the effect of ATP is completely
suppressed by suramin. Activation of CFTR by IBMX (1 mmol/l) and forskolin (2 Amol/l) (I/F) inhibits G
Amil
in the absence or presence of suramin (D) or
hexokinase (5 U/ml) and glucose (15 mmol/l) (E). * indicate significant difference from control. (Number of experiments).
J. Ko
¨
nig et al. / Biochimica et Biophysica Acta 1565 (2002) 17–28 21
guanosine 3V,5V-cyclic monophosphate (cGMP), forskolin,
amiloride, 4,4V-diisothiocyanatostilbene-2,2V-disulfonic acid
(DIDS), hexokinase and naringenin were all from Sigma
(Australia). The peptide-inhibitor of protein kinase G (PKG
inhibitor) H-Arg-Lys-Arg-Ala-Arg-Lys-glu-OH was from
Calbiochem (Australia). Student’s t-test P values < 0.05
were accepted to indicate statistical significance. (Number
of experiments).
3. Results
3.1. Inhibition of ENaC by CFTR and purinergic stimulation
When both CFTR and ENaCs were coexpre ssed in
Xenopus oocytes, an amiloride-sensitive whole cell con-
ductance was detected, which did not show a rundown
during 90-min observation (Fig. 1A). The amiloride-sensi-
tive whole cell current was inhibited upon stimulation of
CFTR by IBMX (1 mmol/l) and forskolin (2 Amol/l). The
inhibition was partially reversible alr eady after 15-min
washout of IBMX and forskolin (Fig. 1B). A complete
recovery of the amiloride-sensitive conductance was
obtained after omitting IBMX and forskolin for 30 min
and deactivation of CFTR. Subsequent restimulation of
CFTR again inhibited G
Amiloride
(Fig. 1C). Thus, the data
indicate a reversible and reproducible inhibition of ENaC
by CFTR in Xenopus oocytes, which cannot be explained
by a rundown of ENaC channel activity. A similar rever-
sible inhibition of amiloride-sensitive transport by CFTR
has been demonstrated in native airway and colonic
epithelia [24,26] and is also show n in Fig. 5 of this paper,
emphasizing on the physiological significance of this
finding.
Fig. 5. (A) Continuous recording of the transepithelial voltage (V
te
) in mouse colon and effects of amiloride on V
te
in the presence or absence of IBMX/
forskolin and hexokinase/glucose. (B) Summary of the amiloride-sensitive short circuit currents (I
sc-Amil
) obtained before and after stimulation of CFTR. I
sc-Amil
is inhibited by CFTR, even in the presence of hexokinase/glucose. Inhibition in the presence of hexokinase/glucose is not different to inhibition of I
sc-Amil
in the
absence of hexokinase/glucose. * indicate significant difference from control (paired t-test). (Number of experiments).
J. Ko
¨
nig et al. / Biochimica et Biophysica Acta 1565 (2002) 17–2822
Similar to CFTR, also purinergic stimulation inhibits
amiloride-sensitive Na
+
transport in the airway epithe-
lium. When stimulated with 100 Amol/l ATP from the
apical side, mouse tracheas responded with a large
increase in the lumen negative transepithelial voltage
(V
te
), indicating transient activation of Cl
secretion
Fig. 6. Transepithelial voltages measured in human colonic biopsies. (A) Continuous recording of the transepithelial voltage (V
te
) in human colonic epithelia
and effects of stimulation by basolateral carbachol (CCH; 100 Amol/l) and luminal UTP (100 Amol/l). Experiments were performed in the continuous presence
of indomethacin and amiloride (both 10 Amol/l). DV
te
= voltage deflection induced by pulsed current injection. (B) Summary of short circuit currents induced
by basolateral CCH and luminal ATP in the absence of stimulation by IBMX/forskolin ( cAMP). (C) Summary of short circuit currents induced by
basolateral CCH and basolateral or luminal ATP in the absence ( cAMP) or after stimulation by IBMX/forskolin ( + cAMP). No effect of ATP on short circuit
currents could be detected. (D,E) Missing effect of stimulation by luminal ATP on I
sc-Amil
. * indicate significant difference from control (paired t-test). (Number
of experiments).
J. Ko
¨
nig et al. / Biochimica et Biophysica Acta 1565 (2002) 17–28 23
(Fig. 2A). In parallel, the effect of amiloride on V
te
and
amiloride-sensitive short circuit currents were largely
inhibited (Fig. 2A,B). The inhibitory effects of purinergic
stimulation on amiloride-sensitive transport were reversi-
ble within 90-min washout of ATP (data not shown). We
further examined the effects of purinergic stimulation on
ENaC in Xenopus oocytes. When applied t o ENaC
expressing oocytes, we did not detect an inhibitory effect
of ATP or UTP on amiloride-sensitive conductance, i.e.
whole cell currents inhibited by amiloride were similar in
the absence or presence of ATP or UTP (Fig. 2C,D).
These experiments exclude the possibility of a direct
inhibitory effe ct of purinergic agonists on ENaC and
indicate the requirement of additional proteins necessary
for inhib ition of ENaC.
3.2. Inhibition of ENaC by CFTR via release of ATP or
UTP?
We examined the possibility that CFTR is controlling the
release of purinergic agonists to the luminal membrane of the
airway epithelium, where they would bind to P2Y receptors,
activate a Ca
2+
-dependent Cl
conductance and inhibit
amiloride-sensitive Na
+
channels. To that end, tracheas were
stimulated with IBMX and forskolin in the presence or
absence of hexokinase (5 U/ml) and glucose (15 mmol/l).
Hexokinase and glucose were applied in order to metabolize
nucleotides, eventually secreted to the luminal side of the
epithelium during activation of CFTR. In case of a substantial
CFTR-controlled release of nucleotides to the luminal side,
we would expect a parallel activation of cAMP and Ca
2+
-
Fig. 7. Effects of cGMP on ENaC expressed in Xenopus oocytes. (A) Continuous recording of the whole cell currents measured in Xenopus oocytes
expressing the epithelial Na
+
channel ENaC, and effects of amiloride (10 Amol/l) (upper trace). Incubation with 1 mmol/l of membrane-permeable cGMP
inhibits ENaC (middle trace). The inhibitory effect of cGMP on ENaC is suppressed by an inhibitor of protein kinase G (lower trace). (B) Summary of the
effects of membrane permeable cGMP on amiloride-sensitive whole cell conductance ( G
Amil
) in the absence or presence of the PKG inhibitor. Note that
cGMP has a slight but significant inhibitory effect on G
Amil
only in the absence of PKG inhibitor. The PKG inhibitor enhanced G
Amil
. (C) Downregulation
of G
Amil
by activation of CFTR with IBMX (1 mmol/l) and forskolin (2 Amol/l) (I/F) takes place in the absence or presence of the PKG inhibitor. # indicate
significantly enhanced G
Amil
in the presence of PKG inhibitor (unpaired t-test). * indicate significant difference from control (paired t-test). (Number of
experiments).
J. Ko
¨
nig et al. / Biochimica et Biophysica Acta 1565 (2002) 17–2824
activated Cl
conductance and thus an attenuated secretion
in the presence of hexokinase and glucose. Although ATP-
induced ion transport was inhibited, IBMX/forskolin (I/F)
activated transport was identical in the presence or absence of
hexokinase/gluc ose (Fig. 3A). Similarly, the purinergic
inhibitor suramin (200 Amol/l) attenuated UTP-induced
transport but had no effect on I/F activated secretion (Fig.
3B). Finally, the blocker of Ca
2+
-activated Cl
channels
DIDS attenuated ATP-induced transport, but was without any
effect on I/F induced secretion (Fig. 3C). Thus, the present
experiments do not deliver any evidence for CFTR-mediated
nucleotide secretion and activation of Ca
2+
-dependent Cl
channels. We therefore suggest that CFTR-mediated inhib-
ition of ENaC is not due to release of ATP/UTP to the luminal
side of the epithelium.
We further examined a possible contribution of nucleo-
tide release to CFTR-mediated inhibition of ENaC in
Xenopus oocytes. To that end, we coexpressed purinergic
P2Y
2
receptors together with ENaC in Xenopus oocytes. As
shown in Fig. 4A, ATP activates a whole cell conductance
in P2Y
2
/ENaC expressing oocytes. However, no inhibition
of the amiloride-sensitive conductance was observed during
stimulation by ATP. In another series of experiments, P2Y
2
receptors and ENaC were coexpressed with CFTR. In this
series of experiments, the transient whole cell conductance
activated by ATP (100 Amol/l) was completely suppressed
by suramin (Fig. 4C). Suramin, however, did not suppress
inhibition of G
Amil
due to activation of CFTR, which again
argues against a CFTR-mediated ATP/UTP release and a
role for inhibition of ENaC. Thus, inhibit ion of G
Amil
was
similar in the absence or presence of suramin (Fig. 4D).
Along this line, hexokinase and glucose did not suppress I/
F-induced inhibition of G
Amil
(Fig. 4E).
The possibility of ATP/UTP release by CFTR in the
intact epithelium was further ruled out. Previous studies
have shown that CFTR inhibits amiloride-sensitive currents
in native human and mouse colonic epithelia [26,42]. In the
present study, we examined whether inhibition of amiloride-
sensitive Na
+
transport by CFTR in mouse colon may
involve secretion of nucleotides. Amiloride-sensitive trans-
port was measured before and after stimulation of CFTR by
IBMX (100 Amol/l) and forskolin (10 A mol/l) and in the
presence of hexokinase (5 mmol/l) and glucose (15 mmol/l).
As shown in Fig. 5, amiloride-induced voltage deflections
were attenuated after stimulation of CFTR and amiloride-
sensitive transport (I
sc-Amil
) was reduced even in the pres-
ence of hexokinase and glucose. Additional experiments
were performed in human colonic biopsies. A previous
study has demonstrated inhibition of I
sc-Amil
by CFTR in
the human colon [26]. Since little is known about the effects
of purinergic stimulation in this tissue, we examined the
impact of luminal and basolateral ATP (100 Amol/l) on ion
transport. The experiments were performed in the presence
of amiloride, after deactivating luminal CFTR Cl
channels
by inhibiting prostaglandin synthesis with indomethacin
[28], or after stimulation of CFTR by IBMX and forskolin.
After inactivation of CFTR, increase of intracellular Ca
2+
by stimulation of basolateral muscarinic M3 receptors with
carbachol (CCH; Amol/l) activates a luminal K
+
secretion.
Similar has been reported previously [25,28,41]. In contrast
to CCH, luminal ATP had no effect on transepithelial
voltage and ion transport in the human colon (Fig. 6A,B).
Moreover, neither luminal nor basolateral ATP showed
effects after stimulation of CFTR ( + cAMP), while stimu-
lation with CCH induced a Cl
secretion. Finally, amilor-
ide-sensitive transport was not inhibited by stimulation with
luminal ATP (Fig. 6D,E). Taken together, these results
suggest that in contrast to the murine colon [18], the human
colonic epithelium does not express purinergic receptors.
Thus, the inhibitory effects of CFTR on ENaC in the human
colon cannot be explained by release of ATP or UTP.
3.3. cGMP and NDPK do not participate in the inhibition of
ENaC by CFTR
Other ATP-dependent processes may play a role for the
inhibition of ENaC. The ATP and ion regulated dependent
NDPK has been shown to be expressed in airway epithelial
cells, where it may interfere with the ion transport
Fig. 8. (A) Continuous recording of the transepithelial voltage (V
te
)in
mouse trachea and effects of amiloride on V
te
in the presence or absence of
membrane permeable cGMP. (B) Summary of the amiloride-sensitive short
circuit currents (I
sc-Amil
) obtained before and application of membrane
permeable cGMP. (Number of experiments).
J. Ko
¨
nig et al. / Biochimica et Biophysica Acta 1565 (2002) 17–28 25
[29,31,39]. Mor eover, it has been shown recently tha t
NDPK binds to and is regulated by adenosine monophos-
phate dependent kinase (AMPK), which is a CFTR-associ-
ated protein defective in CF [40]. Upon activation of NDPK,
ATP is metabolized to UTP or GTP, whi ch could contribute
to the formation of cGMP and inhibit ENaC [5]. This model
is further supported by the fact that both activation of NDPK
and inhibition of ENaC by CFTR are Cl
-dependent
processes [14,31]. We examined the effects of cGMP on
amiloride-sensitive Na
+
currents in oocytes. As shown in
Fig. 7, the amiloride-sensitive Na
+
current is inhibited
slightly but significantly in the presence of 1 mmol/l
membrane-permeable cGMP (Fig. 7A,B). Inhibition of
ENaC by cGMP was abolished by injecting an inhibitor
of PKG. Interestingly, amiloride-sensitive whole cell con-
ductances were enhanced in oocytes inje cted with PKG
inhibitor, whi ch may suggest a basic inhibition of ENaC by
PKG in Xenopus oocytes. However, an inhibitory effect of
cGMP on amiloride-sensitive transport could not be
detected in mouse trachea (Fig. 8). Moreover, the down-
regulation of ENaC by CFTR was undisturbed by the
presence of the PKG inhibitor (Fig. 7C). In addition, the
inhibition of ENaC by CFTR was examined in oocytes,
which had been injected with naringenin (final concentra-
tion 500 Amol/l), a blocker of NDPK, or was demonstrated
in oocytes that coexpre ssed NDPK (Fig. 9A,B). Neither
naringenin nor NDPK interfered with the downregulation of
ENaC by CFTR. In further experiments, we tested the
effects of fisetin and naringenin on mouse airways, but
did not find any significant effects on Cl
secretion or Na
+
absorption (data not shown). Taken together, the present
results do not support a role of either CFTR-mediated
release of ATP, or ATP-dependent NDPK for the inhibition
of ENaC by CFTR.
4. Discuss ion
4.1. ENaC is inhibited by CFTR and by purinergic
stimulation
Previous results and our present result s indicate that
amiloride-sensitive Na
+
transport is inhibited by both
CFTR and purinergic stimulation in native epithelial tissues
[2,24,26,27]. When coexpressed in Xenopus oocytes, ENaC
is inhibited during activation of CFTR. As shown in this
study, downregulation of ENaC by CFTR is reproducible in
the same oocyte and is not due to ENaC channel rundown.
However, in contrast to inhibition by CFTR, purinergic
stimulation does not inhibit ENaC in Xenopus oocytes.
Even in oocytes expressing additional purinergic P2Y
2
receptors, thus clearly demonstrating ATP/UTP-activated
whole cell Cl
currents, inhibition of ENaC by ATP or
UTP was not observed. Obviously, in Xenopus oocytes, a
regulatory element is missing, necessary for inhibition of
ENaC. As another explanation, activation of P2Y
2
receptors
with the subsequent signaling pathway might be too tran-
sient in oocytes, especially when compared to the airway
epithelium. As shown previously, the downregulation of
amiloride-sensitive short circuit currents in mouse trachea
requires 5 10 min of continuous stimulation with ATP or
UTP and a longer lasting activation of Ca
2+
-activated Cl
currents [20]. In the present study, we did not dire ctly
measure ATP release by either epithelial cells or oocytes.
Instead, we performed a functional analysis of the effects of
purinergic stimulation in various tissues and compared the
effects of ATP/UTP on Cl
conductance and amiloride-
sensitive Na
+
absorption. Although any conclusions regard-
Fig. 9. Summary of amiloride-sensitive whole cell conductances ( G
Amil
)
measured in Xenopus oocytes coexpressing ENaC and CFTR. (A) Oocytes
were injected with the inhibitor of nucleoside diphospha te ki nase,
naringenin (final oocyte concentration 50 Amol/l), or water. Down-
regulation of G
Amil
by activation of CFTR with IBMX (1 mmol/l) and
forskolin (2 Amol/l) (I/F) was not suppressed by naringenin. (B) Additional
coexpression of NDPK did not affect downregulation of G
Amil
by activation
of CFTR with IBMX and forskolin. Inhibition of G
Amil
was reversible upon
replacement of extracellular Cl
by gluconate. * indicate significant
difference from control (paired t-test). (Number of experiments).
J. Ko
¨
nig et al. / Biochimica et Biophysica Acta 1565 (2002) 17–2826
ing ATP/UTP release are indirect, the data obtained here do
not supply any evidence for CFTR-dependent nucleotide
release and do not indicate a role for ATP/UTP in inhibition
of ENaC by CFTR in Xenopus oocytes.
4.2. No evidence for release of ATP and activation of CaCC
during stimulation of CFTR
We sought for functional evidence of ATP release, not
by measuring ATP release directly but by detecting activa-
tion of CaCC during stimulation of CFTR in mouse
trachea. Although a recent study demonstrated very low
expression of CFTR in mouse trachea [34], our experi-
ments show activation of Cl
secretion by CFTR, as
indicated by cAMP-induced negative voltage deflection.
Activation of short circuit currents by cAMP, however, was
not affected by hexokinase, a blocker of purinergic recep-
tors (suramin), or Ca
2+
-activated Cl
channels (DIDS).
This argues against any contribution of purinergic stimu-
lationtoCFTR-mediatedsecretioninmousetrachea.
Therefore, our data are in agreement with previously
published reports, which deny a role of CFTR in luminal
ATP release in the airway epithelium [7,22,23,33,38,43].
The same holds true for the mouse intestinal epithelium.
Here, cAMP (IBMX and forskolin) activated secretion and
inhibition of amiloride-sensitive transport was not affected
by hexoki nase treatment. Finally, CFTR-dependent inhib-
ition of Na
+
absorption described for the human intestinal
epithelium [26] is unrelated to ATP/UTP release since no
evidence was found in the present study for expression of
purinergic receptors in the human large intestine and no
effects of ATP on I
sc-Amil
could be detected. Taken
together, the present study does not support the idea of
an autocrine release of ATP by CFTR and inhibition of
amiloride-sensitive currents via this mechanism [36].
4.3. Inhibition of ENaC by CFTR is not due to NDPK
function or cGMP-dependent inhibition
Regulation of ENaC by CFTR has been studied exten-
sively and recent results indicated that ENaC might be
inhibited through the CFTR Cl
current and a change in
the intracellular Cl
concentration [14]. This asks for the
contribution of additional Cl
-sensitive proteins to the
inhibition of ENaC. A Cl
-dependent mechanism has been
reported for the fetal rat alveolar epithelium and the feed-
back inhibition of ENaC in salivary duct cells [4,13,30].In
salivary epithelial cells, aGo and aGi2 proteins mediate the
inhibition of ENaC via increase in intracellular Na
+
and
Cl
, respectively [3,4,13,30]. However, a contribution of
these G protein subunits to the Na
+
feedback or CFTR-
dependent regulation of ENaC in oocytes has not been
found [10,17]. In the present study, we examined the
possible role of NDPK, another Cl
-and cation-dependent
enzyme. NDPK is expressed in the airway epithelium and
is regulated by AMPK, which has been shown to be a
CFTR-associated protein being defective in c ystic fibrosis
[31,39,40]. NDPK has been suggested to play a role in the
control of ion channels in the airway epithelium [31,39]
and thus could be the protein that is in charge of CFTR-
mediated inhibition of ENaC. The data shown here, how-
ever, do not support the idea of NDPK, being the Cl
-
sensitive protein in charge of inhibition o f ENaC in airways
or oocytes. Moreover, although cGMP- and/or cGMP-
dependent PKG may inhibit ENaCs in some cell types
such as Xenopus oocytes or murine nasal epithelium, this
regulatory loop does not affect the control of ENaC by
CFTR [5]. Taken together, it is unlikely that a release of
ATP or UTP contributes to the inhibition of ENaC by
CFTR. This, however, should have been expected if CFTR
shows a significant nucleotide transport. Other ATP-
dependent signaling pathways such as via NDPK or cGMP
are also unlikely to play a role. Since Cl
was found to be
crucial for the inhibition of ENaC, current studies examine
if Cl
ions directly inhibit Na
+
transport through ENaC.
Preliminary results suggest that N or C termini of ENaC
might not be required for the inhibition by CFTR, which
suggests a possible direct inhibition by Cl
ions [42].
Acknowledgements
This work was supported by DFG Ku756/4-1, German
Mukoviszidose e.V., ARC grant 00/ARCS243 and Cystic
Fibrosis Australia.
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    • "Evidence came from impaired cell volume regulation in intestinal crypt epithelia of cystic fibrosis mice (Gawenis et al. 2003; Valverde et al. 1995) and recently from hypoxic myocardial tissue, in which CFTR reduced necrotic cell swelling (Uramoto et al. 2012). It was proposed that CFTR affects volume regulation by autocrine ATP release, although the concept of ATP permeability by CFTR had been rejected in several previous studies (Braunstein et al. 2001; König et al. 2002). In the 4Enhanced apoptotic whole-cell Cl − currents in cells expressing ANO6, ANO9, and ANO10. "
    [Show abstract] [Hide abstract] ABSTRACT: A remarkable feature of apoptosis is the initial massive cell shrinkage, which requires opening of ion channels to allow release of K+, Cl−, and organic osmolytes to drive osmotic water movement and cell shrinkage. This article focuses on the role of the Cl− channels LRRC8, TMEM16/anoctamin, and cystic fibrosis transmembrane conductance regulator (CFTR) in cellular apoptosis. LRRC8A-E has been identified as a volume-regulated anion channel expressed in many cell types. It was shown to be required for regulatory and apoptotic volume decrease (RVD, AVD) in cultured cell lines. Its presence also determines sensitivity towards cytostatic drugs such as cisplatin. Recent data point to a molecular and functional relationship of LRRC8A and anoctamins (ANOs). ANO6, 9, and 10 (TMEM16F, J, and K) augment apoptotic Cl− currents and AVD, but it remains unclear whether these anoctamins operate as Cl− channels or as regulators of other apoptotic Cl− channels, such as LRRC8. CFTR has been known for its proapoptotic effects for some time, and this effect may be based on glutathione release from the cell and increase in cytosolic reactive oxygen species (ROS). Although we find that CFTR is activated by cell swelling, it is possible that CFTR serves RVD/AVD through accumulation of ROS and activation of independent membrane channels such as ANO6. Thus activation of ANO6 will support cell shrinkage and induce additional apoptotic events, such as membrane phospholipid scrambling.
    Full-text · Article · Jun 2016
    • "Stimulation of purinergic receptors by ATP or activation of CFTR stimulate chloride secretion and inhibit amiloride-sensitive Na + transport (ENaC). Inhibition of ENaC by nucleotides is not via CFTR-mediated ATP release [62]. ATP or UTP acting at P2YRs also activate Ca 2+ -dependent Cl -channels. "
    [Show abstract] [Hide abstract] ABSTRACT: Secretomotor reflexes in the gastrointestinal (GI) tract are important in the lubrication and movement of digested products, absorption of nutrients, or the diarrhea that occurs in diseases to flush out unwanted microbes. Mechanical or chemical stimulation of mucosal sensory enterochromaffin (EC) cells triggers release of serotonin (5-HT) (among other mediators) and initiates local reflexes by activating intrinsic primary afferent neurons of the submucous plexus. Signals are conveyed to interneurons or secretomotor neurons to stimulate chloride and fluid secretion. Inputs from myenteric neurons modulate secretory rates and reflexes, and special neural circuits exist to coordinate secretion with motility. Cellular components of secretomotor reflexes variably express purinergic receptors for adenosine (A1, A2a, A2b, or A3 receptors) or the nucleotides adenosine 5'-triphosphate (ATP), adenosine diphosphate (ADP), uridine 5'-triphosphate (UTP), or uridine diphosphate (UDP) (P2X(1-7), P2Y(2), P2Y(4), P2Y(6), P2Y(12) receptors). This review focuses on the emerging concepts in our understanding of purinergic regulation at these receptors, and in particular of mechanosensory reflexes. Purinergic inhibitory (A(1), A(3), P2Y(12)) or excitatory (A(2), P2Y(1)) receptors modulate mechanosensitive 5-HT release. Excitatory (P2Y(1), other P2Y, P2X) or inhibitory (A(1), A(3)) receptors are involved in mechanically evoked secretory reflexes or "neurogenic diarrhea." Distinct neural (pre- or postsynaptic) and non-neural distribution profiles of P2X(2), P2X(3), P2X(5), P2Y(1), P2Y(2), P2Y(4), P2Y(6), or P2Y(12) receptors, and for some their effects on neurotransmission, suggests their role in GI secretomotor function. Luminal A(2b), P2Y(2), P2Y(4), and P2Y(6) receptors are involved in fluid and Cl(-), HCO(3) (-), K(+), or mucin secretion. Abnormal receptor expression in GI diseases may be of clinical relevance. Adenosine A(2a) or A(3) receptors are emerging as therapeutic targets in inflammatory bowel diseases (IBD) and gastroprotection; they can also prevent purinergic receptor abnormalities and diarrhea. Purines are emerging as fundamental regulators of enteric secretomotor reflexes in health and disease.
    Full-text · Article · Aug 2008
  • [Show abstract] [Hide abstract] ABSTRACT: Both stimulation of purinergic receptors by ATP and activation of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibit amiloride-sensitive Na+ transport and activate Cl- secretion. These changes in ion transport may well affect cell volume. We therefore examined whether cell shrinkage or cell swelling do affect amiloride-sensitive Na+ transport in epithelial tissues or Xenopus oocytes and whether osmotic stress interferes with regulation of Na+ transport by ATP or CFTR. Stimulation of purinergic receptors by ATP/UTP or activation of CFTR by IBMX and forskolin inhibited amiloride-sensitive transport in mouse trachea and colon, respectively, by a mechanism that was Cl- dependent. When exposed to a hypertonic but not hypotonic bath solution, amiloride-sensitive Na+ transport was inhibited in mouse trachea and colon, independent of the extracellular Cl- concentration. Both inhibition of Na+ transport by hypertonic bath solution and ATP were additive. When coexpressed in Xenopus oocytes, activation of CFTR by IBMX and forskolin inhibited the epithelial Na+ channel (ENaC) in a Cl- dependent fashion. However, both hypertonic and hypotonic bath solutions showed only minor effects on amiloride-sensitive conductance, independent of the bath Cl- concentration. Moreover, CFTR-induced inhibition of ENaC could be detected in oocytes even after exposure to hypertonic or hypotonic bath solutions. We conclude that amiloride-sensitive Na+ absorption in mouse airways and colon is inhibited by cell shrinkage by a mechanism that does not interfere with purinergic and CFTR-mediated inhibition of ENaC.
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