Prostanoids stimulate K secretion and Cl secretion in guinea
pig distal colon via distinct pathways
Dan R.
Halm and
Susan
Troutman
Halm
Department of Physiology and Biophysics, Wright State University,
Dayton, Ohio 45435
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ABSTRACT |
Short-circuit current
(Isc) and transepithelial conductance
(Gt) were measured in guinea pig distal colonic
mucosa isolated from submucosa and underlying muscle layers.
Indomethacin (2 µM) and NS-398 (2 µM) were added to suppress
endogenous production of prostanoids. Serosal addition of
PGE2 (10 nM) stimulated negative Isc
consistent with K secretion, and concentrations >30 nM stimulated positive Isc consistent with Cl secretion.
PGE2 also stimulated Gt at low and
high concentrations. Dose responses to prostanoids specific for EP
prostanoid receptors were consistent with stimulating K secretion
through EP2 receptors, based on a rank order potency (from
EC50 values) of PGE2 (1.9 nM) > 11-deoxy-PGE1 (8.3 nM) > 19(R)-hydroxy-PGE2 (13.9 nM) > butaprost
(67 nM) > 17-phenyl-trinor-PGE2 (307 nM)
sulprostone (>10 µM). An isoprostane, 8-iso-PGE2,
stimulated K secretion with an EC50 of 33 nM. Cl secretory
response was stimulated by PGD2 and BW-245C, a DP
prostanoid receptor-specific agonist: BW-245C (15 nM) > PGD2 (30 nM) > PGE2 (203 nM). Agonists
specific for FP, IP, and TP prostanoid receptors were ineffective in
stimulating Isc and Gt at
concentrations <1 µM. These results indicate that PGE2
stimulated electrogenic K secretion through activation of EP2 receptors and electrogenic KCl secretion through
activation of DP receptors. Thus stimulation of Cl secretion in vivo
would occur either via physiological concentrations of PGD2
(<100 nM) or pathophysiological concentrations of PGE2
(>100 nM) that could occur during inflammatory conditions.
prostaglandin E2; prostaglandin D2; isoprostane; inflammation
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INTRODUCTION |
FLUID SECRETION IN THE
INTESTINES promotes digestion by dispersing the contents for
access to absorptive sites and for propulsion toward more distal
locations. Excessive fluid secretion increases luminal transit, which
limits absorption and leads to loss of body fluid. Active ion secretion
drives this production of fluid, such that regulatory pathways acting
on ion transporters in secretory cells control the rate of fluid
secretion (19, 20). Prostanoids are powerful stimulators
of ion secretion, producing high, sustained rates across colonic
epithelial cells. Electrogenic secretion of both Cl and K is stimulated
in guinea pig and rabbit distal colon by PGE2 at high
concentrations (>100 nM). Colonic epithelial cells produce this KCl
secretion by an electrogenic mechanism similar to that found in other
fluid secretory epithelia (19, 20, 24, 25). Active K
secretion can be stimulated in the absence of active Cl secretion by
epinephrine (20, 43), aldosterone (21), and
low concentrations of PGE2 (<30 nM) (43).
Thus not only the rate but also the ionic composition of secreted fluid can be controlled by variations in secretory stimuli.
Intestinal inflammation brought on by infection or idiopathic
conditions such as ulcerative colitis occurs with elevated levels of
PGE2 (26, 32). Consequent stimulation of Cl
secretion leads to fluid secretion and symptoms of diarrhea.
PGE2, however, is just one of a large number of compounds
released for signaling by cells in the mucosa. This intercellular
communication is necessary to coordinate various functions including
fluid transport, mucus secretion, muscle contraction, blood flow, as
well as immune recognition and defense (8). Fluid
secretion driven by ion flows serves a general function of limiting
residence of infectious agents in the intestinal lumen, but extreme
rates may result from inappropriate levels of stimulators that occur
during acute responses. The extent of secretory stimulation that
results from pathophysiological signaling has not been determined fully.
Elucidation of secretory regulation in colonic epithelia has been
confounded by the presence of neural elements and immune system
components within the mucosa that can release signaling molecules in
response to diverse stimuli (8). Neural involvement has
been demonstrated by direct nerve stimulation, inhibition of nerve
conduction with tetrodotoxin, or synaptic interference with blockers
such as atropine and
-conotoxins (2, 4, 13, 16, 17, 28, 48,
49). Several extracellular signaling molecules have been shown
to act through stimulating production of prostanoids, generally
PGE2 (4, 7, 44, 53). Routinely this connection
is implicated by using compounds such as indomethacin to inhibit
cyclooxygenase (COX) that leads to synthesis of prostanoids. Other
studies have limited the involvement of extraepithelial elements by
dissection that maintains an intact epithelium so that transepithelial
flow can be measured while several ancillary cell types are removed
(2, 7, 13, 18, 30, 44). In particular, removal of muscle
layers and submucosa largely eliminates influences of enteric nerves on
ion transport (13, 18, 30).
Arachidonic acid can be converted to prostanoids through the action of
COX and specific synthases (10, 38), producing PGD2, PGE2, PGF2
,
PGI2 (prostacyclin), and TxA2 (thromboxane). Receptors selectively responsive to each of these prostanoids have been
identified: DP, EP, FP, IP, and TP, respectively (38). Prostanoid EP receptors constitute a group of four distinct genes (EP1, EP2, EP3, and
EP4), giving a total of eight presently known prostanoid
receptors. Although prostanoid receptors generally interact with one of
the five major prostanoid types with EC50 values of
1-10 nM (1, 5, 31), most of these receptors also have
significant affinity for other prostanoids. Cross-sensitivity of these
receptors at high agonist concentrations (>100 nM) is one reason that
prostanoid responses often have been difficult to characterize.
The study reported here used isolated mucosa from guinea pig distal
colon to establish secretory influences of prostanoids at the
epithelium. Previous measurements of unidirectional isotopic fluxes
(43) demonstrated that Cl and K secretion account
quantitatively for stimulation of short-circuit current
(Isc) by PGE2. Pharmacologically defined prostanoid derivatives provided a means to distinguish activation via various prostanoid receptor subtypes. The results demonstrated that PGE2 stimulated K secretion at
concentrations <100 nM by activating the prostanoid receptor
EP2 subtype. In addition, PGE2 stimulated
electrogenic KCl secretion at concentrations >100 nM, likely through
activation of the prostanoid receptor DP subtype, such that
PGD2 would be a physiological stimulator of colonic Cl secretion.
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METHODS |
Male guinea pigs (400-600 g body wt) received standard
guinea pig chow and water ad libitum. Guinea pigs were killed by
decapitation in accordance with a protocol approved by the Wright State
University Institutional Laboratory Animal Care and Use Committee.
Distal colon was removed and defined as the ~20-cm-long segment
ending roughly 5 cm from the rectum. Colonic segments were cut open
along the mesenteric line and flushed with ice-cold Ringer solution to
remove fecal pellets. Epithelium was separated from underlying submucosa and muscle layers using a glass slide to gently scrape along
the length of the colonic segment. The plane of dissection occurred at
the base of crypts such that only components of the mucosa immediately
adherent to the epithelium remained. Four mucosal sheets from each
animal were mounted in Ussing chambers with an aperture of 0.64 cm2. These sheets were supported on the serosal face by
Nuclepore filters (Whatman), with a thickness of ~10 µm and a pore
diameter of 5 µm. Bathing solutions (10 ml) were circulated by gas
lift through water-jacketed reservoirs that were maintained at 38°C. Standard Ringer solution contained (in mM) 145 Na+, 5 K+, 2 Ca2+, 1.2 Mg2+, 125 Cl
, 25 HCO
, 4 H(3-x)PO
, and 10 D-glucose. Solutions were continually gassed with 95%
O2-5% CO2, which maintained solution pH at
7.4.
Chambers were connected to automatic voltage clamps (Physiologic
Instruments, San Diego, CA) that permitted continuous measurement of
Isc and compensation for solution resistance.
Transepithelial electrical potential difference was measured by two
calomel electrodes connected to the chambers by Ringer-agar bridges.
Current was passed across the tissue through two Ag-AgCl electrodes
connected by Ringer-agar bridges. Isc is
referred to as positive for current flowing across the epithelium from
the mucosal side to the serosal side. Transepithelial conductance
(Gt) was measured by recording currents
resulting from bipolar square voltage pulses (10 mV, 3-s duration)
imposed across the mucosa at 1-min intervals.
Indomethacin, NS-398,
[1S-[1
,2
(z),3
(1E,3S*),4
]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl-5-heptenoic acid (I-BOP), and other prostanoids were obtained from Cayman Chemical (Ann Arbor, MI). SC-51322 was obtained from BioMol (Plymouth Meeting, PA). TTX was obtained from Alomone Labs (Jerusalem, Israel). Butaprost was a generous gift from Dr. H. Kluender of Bayer
Corporation. All other chemicals were obtained from Sigma Chemical (St.
Louis, MO). Drugs were added in small volumes from concentrated stock solutions. Bumetanide, indomethacin, NS-398, and prostanoid derivatives were prepared in ethanol stock solutions. Together indomethacin and
NS-398 resulted in a 0.1% (vol/vol) addition of ethanol, prostanoid derivatives at 10 µM added 0.1% ethanol, and bumetanide addition increased ethanol to 1%. Additions of 1% ethanol alone did not significantly alter Isc or
Gt in basal or secretory states.
Dose responses of Isc and
Gt to prostanoids were fit to
Henri-Michaelis-Menten binding curves using a nonlinear least-squares procedure. Prior findings with guinea pig distal colon indicate that
PGE2 stimulates both negative and positive
Isc components with EC50 values
separated by ~300-fold (43). Those dose responses with more than one
inflection were fit to the sum of two independent binding curves
or
with total Isc or
Gt as a combination of these two components
(IA and IB;
GA and GB) at each
concentration (C). A similar analysis has allowed interpretation of
pharmacological responses to agonists producing two distinct actions
(47). Secretory responses to agonists also were compared
using equivalent electromotive force (EMF) (27, 55)
calculated from the fitted values of Ix and
Gx: EMF = (Ix/Gx). EMF provides a
measure of the active driving force producing electrogenic transport.
Particular transepithelial processes generally produce varied
Isc through the action of a specific transport
EMF even when stimulated by distinct agonists, so that the transport
EMF becomes a useful identifying characteristic of that transport
pathway. Stimulation of Isc can be assessed as
addition of new electrical components in parallel with preexisting basal components, by subtracting basal Isc and
Gt to obtain the stimulated portion. Similarly,
inhibition of Isc would result from deletion of
electrical components. In this manner, action of distinct cell
populations or transport modes can be distinguished according to
intrinsic characteristics of the epithelial transport processes. Time
courses of EMF were calculated by comparing
Isc and Gt with
basal states in which secretory rate was near zero, EMF = (Ix
I0)/(Gx
G0). Results are reported as means ± SE. Statistical comparisons were made using a two-tailed Student's t-test for paired responses, with significant difference
accepted at P < 0.05.
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RESULTS |
Guinea pig distal colonic epithelium spontaneously secretes Cl and
K when mounted in Ussing chambers (43). This secretory activity can be reduced by suppressing prostanoid production with COX
inhibitors such as indomethacin or can be stimulated by adding PGE2 to the bathing solution. Initial
Isc after mounting in Ussing chambers, in the
presence of indomethacin (2 µM), decreased from near zero toward a
negative value approaching
4
µeq · cm
2 · h
1 before
returning to a less negative value (Fig.
1A). Gt
decreased by approximately twofold over this same time interval (Fig.
1B), consistent with reduction of electrogenic ion
transport. Any substances released from isolated mucosa were washed
from the chambers by replacing bathing solution in the reservoirs.
Three washes generally produced maximal change in
Isc and Gt (Fig. 1).
Prostanoid production was suppressed further with a COX-2 inhibitor
(12), NS-398 (2 µM). Equivalent EMF of the
Isc component suppressed by washing and COX
inhibition (Fig. 1C) was similar to the EMF for electrogenic K secretion stimulated by aldosterone (21) or epinephrine
(43). Addition of amiloride (100 µM) to the mucosal
solution inhibited electrogenic Na absorption (Fig. 1) such that
electrogenic transport was in a consistent basal state.

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Fig. 1.
Initial secretory state of isolated colonic mucosa. A
representative time course is shown, starting at initial setup, of
short-circuit current (Isc, A) and
transepithelial conductance (Gt , B)
from guinea pig distal colon epithelium. Indomethacin (2 µM) was
added to both bathing solutions at time 0. Tissues were
washed by draining and replacing the mucosal and serosal bathing
solutions in the reservoirs, producing a ~20:1 dilution of remaining
substances in the tissue chamber. Washing 3 times resulted in
~8,000-fold dilution. NS-398 (2 µM), a selective cyclooxygenase 2 inhibitor, was added to both solutions at ~90 min. Amiloride (100 µM) was added to mucosal solution. Electromotive force (EMF; see
METHODS) of the electrogenic transport (C) that
declined during initial setup period was calculated from
Isc and Gt compared with
values at 100 min; Isc and
Gt after the 3rd wash were too small to
provide a reliable estimate of EMF.
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Addition of PGE2 stimulated both K secretion and Cl
secretion in a concentration-dependent manner (Fig.
2) as reported previously (43). A low concentration of 10 nM produced a large
negative Isc (Fig. 2A) and an
increase in Gt (Fig. 2B), consistent
with stimulation of K secretion. Secretory EMF (Fig. 2C) was
22 mV, similar to the value measured previously for electrogenic K
secretion (21, 43). Subsequent increase of
PGE2 concentration to 3 µM resulted in a positive change
in Isc and further increase in
Gt, consistent with stimulation of Cl secretion.
Although steady-state Isc was near zero,
previous Cl flux measurements (43) indicate that this
change in Isc and Gt
resulted from stimulated Cl secretion in addition to ongoing K
secretion. Blockade of residual nerve activity with TTX (1 µM) or
atropine (10 µM) did not alter the response to PGE2 (data
not shown), similar to observations with mucosal preparations of rat
distal colon (13) and canine proximal colon
(30). Blockade of transmitter release with the combined presence of 300 nM
-conotoxin-GVIA and 300 nM
-conotoxin-MVIIC (
-CgTx), inhibitors of synaptic Ca2+ channels (3,
28, 49), also did not alter the response to PGE2
(data not shown). Addition of bumetanide (100 µM) to the serosal
solution resulted in a positive Isc and a
decrease in Gt, as shown previously to
occur from complete inhibition of K secretion and only partial
inhibition of Cl secretion (43). The large positive EMF
(Fig. 2C) was consistent with continuing Cl secretion in the
absence of K secretion.

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Fig. 2.
Stimulation of electrogenic ion secretion. A
representative stimulation of secretion is shown with
Isc (A), Gt
(B), and EMF (C). Initial condition followed 3 washes (see Fig. 1) and included indomethacin (2 µM), NS-398 (2 µM), and amiloride (100 µM). EMF of stimulated electrogenic
transport was calculated in reference to Isc and
Gt at time 0 (see
METHODS). PGE2 was added to serosal solution:
10 nM at time 0, 3 µM at ~15 min. Bumetanide (100 µM)
was added to serosal solution.
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Activation of sustained ion secretion.
Concentration-related stimulation of K and Cl secretion suggested
independent stimulatory pathways, possibly through actions of
PGE2 at multiple receptors. Because prostanoid receptors of the EP subtype have affinities for PGE2 in the low
nanomolar range (1, 5, 10, 31), sensitivity to stimulation
by agonists with defined affinity for EP receptor subtypes (10,
38) was tested. Secretion was measured from steady-state
Isc and Gt 20 min after
each concentration increase. This time interval was sufficient for
Isc to relax after concentration steps smaller than those shown in Fig. 2. These dose responses of steady-state Isc and Gt (Fig.
3) exhibited complex curvature suggesting
two interactions (47) with stimulatory pathways for K and
Cl secretion. Independent fits of Isc and
Gt to binding curves (see METHODS) produced identical rank order potencies and similar EC50
values for each agonist (Table 1). For
the negative Isc response, the observed rank
order potency of PGE2 > 11-deoxy-PGE1
(11dPGE1) > 19(R)-hydroxy-PGE2
(19hPGE2) > butaprost > 17-phenyl-trinor-PGE2 (17pPGE2)
sulprostone
supports involvement of EP2 receptors. The similarity of
EMF stimulated by these agonists (Table 1 and Fig. 3E)
suggests that the identical transport process was stimulated in each
case. Because the EMF for these responses was similar to the K
secretory EMF (21, 43), the EP2 prostanoid
receptor is likely an initiator of K secretion.

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Fig. 3.
Secretory dose response to EP receptor
agonists. Cumulative dose responses were measured, similar
to Fig. 2, for steady-state Isc (A
and B) and Gt (C and
D). 8-iso-PGE2 is an isoprostane formed
independent of cyclooxygenase activity. Other compounds have defined
affinity for EP prostanoid receptors (Refs. 1,
5, 10, 31, 38):
PGE2 (all), 11-deoxy-PGE1 (EP4,
EP2, EP3),
19(R)-hydroxy-PGE2 (EP2,
EP4), butaprost (EP2),
17-phenyl-trinor-PGE2 (EP1, EP3,
EP2, EP4), sulprostone (EP3,
EP1). Each dose response was fit to the sum of 2 binding
curves (see METHODS), a response at low concentration and a
response at high concentration. These 2 responses consisted of negative
Isc and positive Isc
components representing electrogenic K secretion and Cl secretion,
respectively. Numbers of experiments averaged for each dose response
are listed in Tables 1 and 2. Control and bumetanide-inhibited
conditions are also shown. *Bumetanide-insensitive
Isc significantly different from 0 (P < 0.05) (A and B). Error
estimates for Gt (C and D)
were calculated after subtracting control value for each tissue.
E: current and conductance fit to each component are plotted
(see Tables 1 and 2). Values from negative current ( )
and positive current ( ) responses were fit by least
squares, and slope of line is secretory EMF, 24.7 mV and +13.0 mV,
respectively.
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Stimulation of Cl secretory Isc had
EC50 values in the micromolar range (Fig. 3 and Table
2), suggesting that these agonists also
were interacting with another class of receptor. Agonists selective for
other prostanoid receptors were tested for efficacy in stimulating Cl
secretion: BW-245C for DP, fluprostenol for FP, carbaprostacyclin for
IP, and I-BOP for TP (10, 38). Only BW-245C
stimulated Isc and Gt
significantly, either alone or during K secretion stimulated with 10 nM
PGE2 (Fig. 4). Inhibition of nerve activity with TTX (10 µM) or atropine (10 µM) did not alter steady-state responses to BW-245C or PGD2 (data not shown).
Activation with BW-245C produced a steady-state stimulation similar to
PGE2 (Fig. 4) but only partially reproduced the transient
component of Isc. The similarity of EMF suggests
that the identical transport process produced responses to BW-245C and
high-concentration PGE2 (Fig. 4C). In addition,
the DP agonist alone appeared to stimulate electrogenic KCl secretion
similar to high-concentration PGE2 (Ref. 43;
Fig. 2C), based on EMF (Fig. 4C) and a large
increase in Gt (Fig. 4B). Inhibition
with bumetanide (Fig. 4) produced similar results in paired tissues,
indicating that DP receptors also activated bumetanide-insensitive
secretory Isc. EMF with bumetanide (Fig.
4C) was consistent with Cl secretion remaining after
complete inhibition of K secretion, as observed previously (43).

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Fig. 4.
Secretory stimulation via DP receptors. A representative
stimulation of secretion is shown with Isc
(A), Gt (B), and EMF
(C). Initial condition followed 3 washes and included
indomethacin (2 µM), NS-398 (2 µM), and amiloride (100 µM). At
time 0 (1st stim), low-concentration (10 nM)
PGE2 ( , ) stimulated
electrogenic K secretion, and addition of BW-245C (10 µM;
), a DP receptor agonist (PGD2 analog),
stimulated positive Isc, consistent with Cl
secretion. Subsequent addition (~25 min, 2nd stim) of higher
concentration (10 µM) PGE2 ( ) or BW-245C
( ) converted K secretion to electrogenic KCl secretion,
but low-concentration (10 nM) PGE2 ( ) had
little additional action on BW-245C-stimulated secretion. Bumetanide
(100 µM) was added to serosal solution. EMF of stimulated
electrogenic transport was calculated in reference to
Isc and Gt at time
0 (see METHODS).
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Addition of either PGD2 or BW-245C resulted in
Isc becoming more negative at low concentrations
and more positive at higher concentrations (Fig.
5A), although not as
pronounced nor over as wide a concentration range as with
PGE2. Dose responses of Gt to either
PGD2 or BW-245C could be fit readily with a single binding
curve (Fig. 5B). The small decline in
Isc with either DP agonist may simply reflect
slightly greater sensitivity for activation of K secretion than for Cl
secretion, such that the actual EC50 of the
Isc response would be between these two values (Table 3). For electrogenic KCl
secretion, Gt measurements have the advantage of
not depending on the direction of transepithelial charge flow. Together
these results suggest that PGD2 and BW-245C both acted at
only a single receptor type to produce electrogenic KCl secretion.
EC50 values obtained with Gt
responses (Tables 2 and 3) produced a rank order potency supporting
involvement of DP receptors: BW-245C > PGD2 > PGE2 > butaprost > 11dPGE1 = 19hPGE2 > 8-iso-PGE2 (8iPGE2)
17pPGE2 and carbaprostacyclin
fluprostenol, I-BOP,
and sulprostone. The inability of fluprostenol, I-BOP, or sulprostone
to stimulate Isc or Gt
also underscores that the observed actions of PGE2 were not
a generalized response to prostanoids.

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Fig. 5.
Secretory dose response to DP receptor agonists.
Cumulative dose responses of PGE2, PGD2, and
BW-245C were measured for steady-state Isc
(A) and Gt (B). BW-245C is
a DP receptor-selective agonist (PGD2 analog). Each dose
response was fit to binding curves as in Fig. 3. Responses of
Gt to PGD2 and BW-245C required only
a single binding curve for fitting. In 2 groups ( ,
), PGE2 (10 nM) was added before dose
response. Nos. of experiments averaged for each dose response are
listed in Table 3. Control and bumetanide-inhibited conditions are also
shown. *Bumetanide-insensitive Isc significantly
different (P < 0.05) from PGE2 condition
(A). Error estimates for Gt
(B) were calculated after subtracting control value for each
tissue.
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In an attempt to determine action on Cl secretion independent of graded
K secretory responses, K secretion was stimulated with 10 nM
PGE2 before dose responses with DP agonists (Fig. 5). Both
Isc and Gt responses
could be fit with single binding curves, but the resulting
EC50 values obtained from Isc
responses were larger than from Gt responses
(Table 3). These higher EC50 values for
Isc measurements may reflect a small portion of
stimulated K secretion that still occurs with greater sensitivity than
for Cl secretion, such that positive deflections in
Isc occur at higher concentrations. Because the
specific DP receptor antagonist BWA-868C (10) is no longer
commercially available, a direct test of PGE2 action at DP
receptors was not possible.
Positive Isc was stimulated by PGE2,
PGD2, or BW-245C, in the presence of bumetanide (Fig.
6). Previous flux measurements support
the idea that this PGE2-stimulated
Isc is Cl secretion without any accompanying K
secretion (43). Although PGE2 produced higher
steady-state Isc and Gt,
PGD2 and BW-245C activated with generally lower
EC50 values (Figs. 5 and 6; Table 3). This response to
PGD2 and BW-245C was identical when measured during
stimulation with 10 nM PGE2 (data not shown), indicating
that the larger response by PGE2 likely was not caused by
an additional activation of EP receptors. Addition of 10 µM
PGE2 after stimulation with either PGD2 or
BW-245C (10 µM) increased Isc and
Gt (Fig. 6), consistent with a greater secretory
rate through PGE2 action.

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Fig. 6.
Bumetanide-insensitive secretory response. Cumulative
dose responses of PGE2, PGD2, and BW-245C were
measured for steady-state Isc (A) and
Gt (B) in tissues with bumetanide
(100 µM) present in serosal solution. Each dose response was fit by a
single binding curve, similar to those in Fig. 5. Nos. of experiments
averaged for each dose response are listed in Table 3. Control and
PGE2-stimulated (10 µM) conditions are also shown. For
tissues with PGD2 or BW-245C, increases stimulated by
PGE2 (10 µM) were significantly different from zero
(P < 0.05), as indicated by asterisks. Error estimates
for Gt (B) were calculated after
subtracting control value for each tissue.
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Activation of transient ion secretion.
Previous measurements of secretory activation by prostanoids in guinea
pig distal colon focused on maximal Isc
responses (16, 17, 50). Maximal Isc
response to PGE2 generally was dominated by transient
components, and BW-245C stimulated much smaller transient Isc (Figs. 2 and 4). The positive secretory EMF
during the first 5 min of stimulation (Fig. 4C), together
with previous flux measurements (43), is consistent with
Cl secretion as the source of the transient Isc.
Comparison of Isc responses during concentration
steps of dose responses (Fig. 5) shows that BW-245C and
PGD2 produced steep early rises in
Isc that were small and dominated by the later steady-state plateau (Fig.
7A). EP agonists
11dPGE1 and 19hPGE2 also produced steep early
increases in Isc that were attenuated (Fig.
7B). The time course of stimulation by the EP2
agonist butaprost was much delayed (Fig. 7B), which may have
resulted from slow conversion to the more potent free acid form
(1, 5). The isoprostane 8iPGE2 also did not
produce a noticeable transient Isc response
(data not shown). These results indicate that agonists for DP and EP
prostanoid receptors are relatively weak stimulators of the transient
Isc response.

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Fig. 7.
Transient Isc response to
PGE2. Mucosae from the same colon were stimulated with
prostanoids. Initial condition followed 3 washes and included
indomethacin (2 µM), NS-398 (2 µM), and amiloride (100 µM).
A: PGE2, PGD2, and BW-245C were
added to serosal solution in cumulative dose responses (Fig. 5). Change
in Isc ( current) during step increase from
100 nM to 1 µM is shown at time 0. B:
PGE2, 11dPGE1, and 19hPGE2 were
added to serosal solution in cumulative dose responses (Fig. 3).
Current during step increase from 100 nM to 1 µM is shown at
time 0. Butaprost was added in a cumulative dose response to
a tissue from another colon (Fig. 3). Current during step increase
from 300 nM to 1 µM is shown at time 0. C:
PGE2 was added in the absence or presence of AH-6809 (10 µM, 100 µM) in serosal solution. PGE2 was added to
serosal solution in a cumulative dose response. Current during step
increase from 300 nM to 1 µM is shown at time 0; AH-6809
had been present ~140 min at time of these PGE2
additions. D: Isc during addition of
PGE2 and BW-245C to serosal solution 3 µM is shown in the
presence of serosal solution bumetanide (100 µM). AH-6809 (100 µM)
was added to serosal solution of one tissue. Bumetanide and AH-6809 had
been present ~30 min at time of prostanoid addition.
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Transient components were measured as the difference between maximal
Isc and subsequent steady-state
Isc; because of large variability among
responses at ~1 min (Figs. 2A and 4A), maximal Isc was measured ~2 min into the response.
Also, in a few experiments, TTX (1 µM) reduced peak
Isc stimulated by BW-245C (3 µM) at ~1 min
by ~2 µeq · cm
2 · h
1
without altering latter parts of the response (data not shown). Dose
responses of transient Isc to PGE2
(from experiments in Fig. 5) had an EC50 of roughly 200 nM.
Transient components measured during cumulative dose responses (Fig. 7,
A and B) were much smaller than for a single
large increase in concentration (Figs. 2A and 4A), which may result from desensitization as observed for
prostanoid stimulation of secretory Isc in
rabbit ileum (37). Desensitization could lead to an
overestimation of the EC50 for activation of transient
Isc.
The prostanoid antagonist AH-6809, which has species-dependent
specificity for EP1, EP2, EP3, DP,
and TP prostanoid receptors (1, 10, 38), distinctly
reduced the transient component of the secretory response at a
concentration of 100 but not 10 µM (Fig. 7C). The
PGE2 response in the presence of 100 µM AH-6809 (Fig.
7C) was similar in form to the stimulation by agonists for DP and EP prostanoid receptors in the absence of this inhibitor (Fig.
7, A and B). Bumetanide-insensitive
Isc (Fig. 7D) had a transient
component that was finished within ~2 min, as reported previously
(43), indicating that the broad shoulder of the transient component (Figs. 2A and 4A) was entirely
bumetanide sensitive. In addition, AH-6809 (100 µM) reduced the
steady-state Isc stimulated by PGE2
in the presence of bumetanide by 0.71 ± 0.10 µeq · cm
2 · h
1
(n = 6), similar to the difference between
PGD2 and PGE2 stimulation (Figs. 5A,
6A, and 7D).
Inhibition of transient Isc by AH-6809 only at
high concentration suggests an action via a pathway independent of
prostanoid receptors. Weak stimulation of the transient component by DP
and EP agonists (Fig. 7, A and B) indicates that
neither DP nor EP receptors were primarily involved in this transient
Isc response. Prior stimulation with low
concentration PGE2 did not augment substantially the
transient component produced by BW-245C (Fig. 4A),
indicating further that combined action at DP and EP receptors was not
required to produce this transient response. In addition, sulprostone
(1 µM), an EP3 and EP1 agonist (1, 10,
38), did not augment the transient response with BW-245C (10 µM) when added before stimulation, and SC-51322 (1 µM), an
EP1 antagonist (1), did not reduce the
transient response to PGE2 (1 µM) (data not shown).
Absence of a transient response during butaprost stimulation of Cl
secretion, as well as during 11dPGE1 and
19hPGE2 stimulation, further supports a lack of involvement
by EP2 receptors (Fig. 7B). Similarly,
TP prostanoid receptors were likely not involved in producing the
transient Isc response, because the TP agonist I-BOP (300 nM) did not augment the BW-245C (10 µM) response and the TP
antagonist SQ-29548 (1 µM) (1, 10, 38) did not reduce the PGE2 (1 µM) response (data not shown). Together these
results support the lack of involvement in this transient
Isc response by EP1,
EP2, EP3, DP, and TP prostanoid receptors. Thus
the pharmacological profile of activation and inhibition suggests that
PGE2 did not stimulate transient Cl secretion by activating
known prostanoid receptors but rather acted through a presently
unidentified receptor.
 |
DISCUSSION |
Numerous neurotransmitters and locally produced mediators can
stimulate colonic ion secretion (4, 8, 20). Many of these
agents work through signaling pathways that converge on production of
prostaglandins, which provide the final stimulus to epithelial cells
(8, 44, 50, 53). PGE2 is an effective stimulator of secretion, and this action has been studied extensively. The chief intracellular second messenger appears to be cAMP (20, 37, 51), so a reasonable assumption based on prostanoid receptor characteristics (10, 38) would be that PGE2
acts through EP2 or EP4 receptors. In guinea
pig distal colon, the PGE2 dose response for stimulating
ion secretion ranges over six orders of magnitude (43).
Identification of the receptors involved in activating this
wide-ranging secretory response can be approached more explicitly now
because agonist responses of the eight identified prostanoid receptors
have been characterized.
Prostanoid receptors.
Prostanoid receptors have been classified into eight distinct
pharmacological types (10) corresponding with the major
prostanoid compounds, PGD2, PGE2,
PGF2
, PGI2, and TxA2. These
receptors are the products of eight identified genes and are expressed
in many of the tissues exhibiting specific prostanoid responses
(38). Binding sites for PGE2 have been detected
in rabbit distal colonic crypt epithelial cells with EC50
values of 0.3 and 11 nM (29). All four EP receptors were
detected in colonic epithelium with in situ hybridization of mRNA for
these receptors, although differences between rat (39) and
mouse (36) may be caused by variations in relative
abundance of mRNA among these receptors. In situ hybridization for mRNA
of the DP receptor indicates localization to surface columnar cells of
rat colonic epithelium (54). Interestingly, none of the
knockout mice deficient of prostanoid receptors had dramatic intestinal
dysfunction (46).
Establishing a link between a receptor subtype and a cellular function
can be accomplished by altering the response with specific activators
or inhibitors of the receptors. The presence of the receptor alone
would not indicate a signaling connection to a particular response.
Efficacy of prostanoid derivatives at prostanoid receptor subtypes has
been evaluated recently (1, 5, 31). Binding of
PGE2 indicated an EC50 for EP2
receptors of 5 nM in human (1), 12 nM in mouse
(31), and 7 nM in rat (5). For EP4 receptors, PGE2 EC50 values
were 0.8 nM in human (1), 1.9 nM in mouse
(31), and 1.1 nM in rat (5). For
distinguishing EP subtypes, butaprost is specific for EP2
and has highest potency when deesterified to the free acid form
(1, 5). Other compounds (1, 5, 31) generally
interact with more than one of the EP receptor subtypes and provide
less specific determination of subtype involvement. PGE2
interacts less strongly with DP and FP receptors and only very weakly
with IP and TP receptors; EC50 was 100 nM for human and
mouse FP receptor (1, 31), 300 nM for human DP receptor
(1), and >10 µM for human and mouse IP and TP receptors
(1, 31). These receptor affinities for PGE2 suggest that most physiological actions would be with EP receptors but
that at 10- to 300-fold higher concentrations activation of FP and DP
receptors might occur.
Colonic secretory response.
Secretory activation of distal colonic epithelium by PGE2
consisted of three major components (Fig. 2) that appear to be
initiated by three distinct receptor-linked pathways. Electrogenic K
secretion was stimulated via EP2 prostanoid receptors;
sustained electrogenic KCl secretion was stimulated via DP prostanoid
receptors; and transient electrogenic Cl secretion was stimulated via
an unidentified receptor type.
Electrogenic K secretion requires apical membrane K channels together
with basolateral membrane Na/K pumps, Na-K-2Cl cotransporters, and,
presumably, Cl channels (20, 43). This K secretory
response has the high affinity (EC50 1-3 nM) for
PGE2 expected of EP receptors (Tables 1 and 3). Designation
of the response as EP2 relies primarily on the stimulation
by butaprost. Relatively high butaprost affinity suggests that
esterases in the mucosa converted the terminal methyl ester to a free
acid form, which has higher affinity for EP2 receptors
(1, 5). The inability of sulprostone to stimulate secretion (Fig. 3) strongly supports the absence of involvement of
EP3 and EP1 receptors. Involvement of
EP4 receptors cannot be excluded entirely because of the
lack of specific agonists or antagonists. However, because butaprost
completely reproduced the K secretory response (Table 1),
EP2 receptor activation was sufficient for secretory stimulation.
Electrogenic KCl secretion requires apical membrane K and Cl channels
together with basolateral membrane Na/K pumps, Na-K-2Cl cotransporters,
and K channels (20, 43). Activation of sustained electrogenic KCl secretion via DP receptors was indicated by the ability of PGD2 and BW-245C to stimulate this secretion
(Figs. 4 and 5) with an EC50 lower than that for
PGE2 (Table 3). These DP receptor agonists stimulated
bumetanide-insensitive Isc (Fig. 6), suggesting
that basolateral Cl uptake also could occur via another transport
mechanism to produce Cl secretion.
Transient electrogenic Cl secretion apparently requires apical membrane
Cl channels together with basolateral membrane Na/K pumps, Na-K-2Cl
cotransporters, and K channels. Stimulation occurred at high
PGE2 concentrations but not via activation of any of the defined prostanoid receptors (Fig. 7, A and B).
The high selectivity for PGE2 over other prostanoids,
however, does suggest action through a specific receptor. A requirement
for Na-K-2Cl cotransporters is supported by sensitivity to bumetanide
(Figs. 2A, 4A, and 7D). However, a
limited capacity to produce basolateral Cl entry by means other than
Na-K-2Cl cotransport is supported by the sustained and
bumetanide-insensitive Isc produced by
PGE2 in excess of that produced by BW-245C (Fig.
7D). Thus this so-called transient response to
PGE2 is best characterized as a nonprostanoid receptor
stimulation of Cl secretion with a large bumetanide-sensitive transient
component and a much smaller bumetanide-insensitive sustained component.
All three of these secretory responses appear to involve increases of
intracellular cAMP. Both EP2 and DP prostanoid receptors are linked to stimulation of adenylate cyclase (10, 38)
and forskolin, which activates adenylate cyclase, stimulates a large, partially transient Cl secretory response in guinea pig distal colon
(43). Clearly, cAMP alone could not produce these distinct secretory modes unless each occurs in a separate epithelial cell type.
Although subpopulations of cells in the colonic epithelia with
different receptors are possible, an equally plausible explanation is
that each of these receptors produces multiple intracellular second
messengers that permit variation in the secretory response. Prostanoid
receptor subtypes have been shown to generate more than a single second
messenger (38). In addition, multiple second messengers
may be required simply to coordinate the activity of the channels and
cotransporters necessary to produce any transepithelial ion flow.
Previous measurements of colonic secretory sensitivity to
PGE2 (or PGE1) generally were taken from peak
Isc response and had EC50 values of
50-3,000 nM. Stimulation of Cl secretory
Isc had EC50 values of 50 nM in
canine proximal colon (14, 30, 41), 60 nM in equine
proximal colon (9), ~3,000 nM in guinea pig distal colon
(50), ~150 nM in porcine distal colon (48),
200 nM in rabbit distal colon (35), and ~100 nM in rat
colon (40). Stimulation of K secretion in rabbit distal
colon had an EC50 of 100 nM (35). The
basolateral membrane electrical potential difference of rabbit distal
colonic crypts depolarized during PGE2 addition with an
EC50 of ~100 nM (34). The EC50
obtained did not appear to depend on whether a mucosal or
mucosal/submucosal tissue preparation was used. For the human colonic
cell line T84, stimulation of Cl secretory Isc
had an EC50 of 10-30 nM (51, 52) whereas
cAMP production had an EC50 of 100 nM (51). In light of the high affinity (0.8-12 nM) of EP2 and
EP4 receptors for PGE2 (1, 5, 31),
these colonic Cl secretory responses probably result from activation of
another class of receptor that binds PGE2 with lower affinity.
Two major factors may have contributed to a difficulty in recognizing
the action of stimulation through EP receptors in colonic epithelia:
relatively low rates of K secretion and mucosal production of
prostanoids. Rates of K secretion are generally <1
µeq · cm
2 · h
1 in rabbit,
rat, and human distal colon (20, 44) but ~3
µeq · cm
2 · h
1 in guinea
pig distal colon (43). In addition, concurrent stimulation of electrogenic K and Cl secretion at high PGE2
concentrations obscures the extent of activation when only
Isc measurements are used to quantify the
response. Use of a mucosa preparation largely eliminates secretory
influences from enteric nerves (2, 13, 30) so that
exogenous stimulation can be more easily interpreted. However, colonic
mucosa is capable of producing the five major prostanoids, including
PGE2 (4, 7, 11, 33, 44, 53), so that K
secretion would often be highly stimulated in the initial periods of
many experiments. Even in the presence of indomethacin to suppress
prostanoid production, guinea pig distal colon (Fig. 1) was apparently
stimulated beyond the EC50 value. Only after in situ
stimulators were reduced by rinsing the mucosa were basal secretory
rates low enough to allow for ready detection of stimulation by
concentrations of PGE2 in the range of 0.1-10 nM
(Figs. 2 and 3). Similarly, in human jejunum PGE2
stimulation of Cl secretory Isc occurred with an
EC50 of 1 nM only after suppression of endogenous prostanoids (6). Together, suppression of endogenous
activators and measurements of both Isc and
Gt allow for a more complete view of colonic
secretory responses.
Stimulation of Cl secretion by PGE2 (Fig. 3; Refs.
9, 14, 30, 34,
35, 40, 41, 48,
50) probably involves low-affinity activation of DP or FP
receptors, based on the PGE2 affinity of these receptors
(1, 5, 31). Secretory activation by PGD2 or
PGF2
in guinea pig colon, measured from peak
Isc, was influenced by nerve and COX activity
(16, 17). Stimulation by PGF2
in canine
proximal colon was eliminated by indomethacin (41).
PGD2 inhibited Cl secretion via enteric nerves in rat distal colon (18) and through a PGD2
metabolite in canine proximal colon (41). PGD2
activation of secretion (Fig. 5) probably was not just an alternate way
to stimulate electrogenic KCl secretion; rather, PGE2 acted
through DP receptors because the PGD2 EC50 was
lower than that for PGE2 (Table 3) and consistent with
EC50 values for DP receptors (1, 5, 31).
Transient Cl secretion apparently occurred via nonprostanoid receptors
with high selectivity for PGE2. A similar low-affinity
stimulation of Cl secretory Isc occurs in canine
proximal colon (42).
Use of a mucosa preparation and blockade of COX with indomethacin and
NS-398 in the present study indicate that the observed secretory
activation (Fig. 2) did not occur through release of another prostanoid
and support a lack of enteric nerve involvement. Although the three
secretory responses of PGE2 were likely produced via
epithelial receptors, stimulation through another cell type remaining
in the mucosa cannot be absolutely excluded. However, any response
acting through mucosal nerve processes would have to occur without
action potential propagation (TTX insensitive) or neurotransmitter
release (
-CgTx insensitive and atropine insensitive). Because EP and
DP receptors are present on epithelial cells (36, 39, 54)
and most stimulatory pathways appear to converge on prostanoid release
(8), an epithelial location for these secretion-initiating receptors is consistent with current understanding of mucosal functions.
Bumetanide-insensitive secretory Isc has not
been as well characterized as bumetanide-sensitive Cl secretion, but it
appears to consist of electrogenic Cl secretion and, depending on the species, a small component of HCO3 secretion (43,
45). In guinea pig distal colon, bumetanide-insensitive
Isc is dependent on Cl and HCO3 and
is insensitive to hydrochlorothiazide and disulfonic stilbenes (43), so
the mechanism of basolateral Cl uptake remains unclear. The stimulation
of bumetanide-insensitive Isc by
PGD2 and BW-245C (Fig. 6; Table 3) indicates that DP
receptors activated bumetanide-insensitive as well as
bumetanide-sensitive Cl secretion. The nonprostanoid receptor response
also includes a small component of bumetanide-insensitive
Isc (Figs. 6A and 7D) in
addition to the large bumetanide-sensitive transient
Isc component (Fig. 4A).
Inflammatory conditions.
Stimulation of Cl secretion in the colon can be accomplished through
activation of several receptor-coupled pathways (8, 20).
PGE2-mediated stimulation in epithelial cells apparently involved DP prostanoid receptors. Because both DP and EP2
receptors can initiate increases of intracellular cAMP
(38), involvement of these two receptor types in secretory
activation is consistent with increased intracellular cAMP during
PGE2 addition (51) and the ability of
forskolin or theophylline to produce secretion (19, 20).
Other intracellular second messengers probably are involved in each of
these receptor-initiated events (38). Elevation of
PGE2 concentration occurs during various conditions such as bacterial infection, laxative treatment, irritable bowel syndrome, and
ulcerative colitis (26, 32).
Measured PGE2 ranges over several orders of magnitude
depending on the state of the tissue. In isolated mucosa of rabbit
distal colon (7), basal levels were in the range of
0.5-2 nM and increased to 20-40 nM during stimulation with
arachidonic acid or the calcium ionophore A-32187. Rat colonic
epithelial cells generated PGD2, PGE2,
PGF2
, 6-keto-PGF1
(PGI2
metabolite), and TxB2 (TxA2 metabolite) in
roughly similar proportions, although substrate availability may alter
the relative production of these prostanoids (11, 33).
Luminal dialysates (32) were ~1 nM in colon of healthy
humans and were modestly elevated for individuals with Crohn's colitis
(5 nM) or Clostridium difficile colitis (3 nM), whereas levels in ulcerative colitis patients were distinctly elevated
(44 nM). Prostanoid concentrations near the epithelial cells
probably were higher, so levels in healthy individuals might stimulate
electrogenic K secretion through EP2 receptors.
Pathophysiological conditions would produce higher PGE2
levels that presumably could lead to sustained electrogenic KCl
secretion via DP receptors and transient Cl secretion via an
unidentified eicosanoid receptor.
Isolation of colonic mucosa for in vitro measurement of
Isc and Gt (Fig. 1) can
be viewed as an inflammatory response, because the tissue tearing that
separates mucosa from submucosa undoubtedly stimulates production of
numerous compounds including eicosanoids such as PGE2. In
this context, the initial time course of Isc and
Gt can be seen as a waning of the stimulation
produced acutely by inflammatory mediators. With dose responses to
PGE2 (Fig. 3), changes in Isc and
Gt (Fig. 1) can be interpreted as effective PGE2 concentration at secretory epithelial cells in vitro.
Initial effective PGE2 concentration would be ~1 µM
(Fig. 1), falling rapidly over the first 10 min to ~10 nM and
stabilizing after ~30 min at ~4 nM. Estimating mucosal volume as
~30 µl (0.64-cm2 area and ~500-µm thickness), the
~300-fold dilution into the serosal bathing solution of released
inflammatory mediators was comparable to the ~250-fold drop in
effective PGE2 concentration; PGE2 release
occurs predominantly at the serosal side (6). The first
wash dropped effective PGE2 concentration further to ~1
nM, which was followed by a drop to ~0.4 nM after the second wash and
to ~0.3 nM after the third wash. Although these values overestimate
PGE2 concentration by assuming that the stimulation resulted only from endogenous PGE2, the final estimated
level is just below the range for in vitro PGE2
measurements (~1-2 nM) from similarly isolated human and rabbit
distal colonic mucosa (7, 44).
Although many control pathways ultimately can produce fluid secretion
across colonic epithelia (8), results from this study indicate that of the prostanoid receptors only EP2 and DP
subtypes are likely to be coupled directly for activation of sustained electrogenic K and Cl secretion in secretory epithelial cells. The
consequences of these multiple epithelial receptors are that fluid
secretory rate and composition can be adjusted more precisely than if a
single pathway is used to initiate secretion. Activation of
EP2 receptors would produce a primary secretion of K that
creates a lumen positive electrical potential difference driving
passive Cl secretion. The fluid produced would have relatively high K concentrations that may contribute to the barrier function of the
epithelium. Activation of DP receptors would produce active Cl
secretion together with some proportion of K secretion, depending on
the species. The amount of K secretion relative to Cl secretion in
distal colon varies from roughly equal in guinea pig (43) to ~25% of Cl secretion in rabbit (20) and ~10% in
human (44). Mucosal mast cells can release
PGD2 (15), so this activation of electrogenic
KCl secretion could begin either from an epithelial (11,
33) or an extraepithelial (11, 15, 33) signal. In
addition, high concentrations of PGE2 (2-5 µM)
stimulated mucus as well as fluid secretion in human colonic crypts
(22); although the nature of the receptor involved was not
determined, fluid secretion was augmented by addition of macromolecules
that presumably serve barrier functions of the epithelium. Thus low
levels of PGE2 would produce a fluid of higher K
concentration, whereas pathophysiological levels of PGE2,
generated acutely in response to infection or other inflammatory
condition, would produce larger fluid secretion with relatively lower K concentrations.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-39007.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: D. R. Halm, Dept. of Physiology and Biophysics, Wright State University, 3640 Colonel Glenn Hwy., Dayton, OH 45435 (E-mail:
dan.halm{at}wright.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 25 January 2001; accepted in final form 4 June 2001.
 |
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