Centro de Biofísica y Bioquímica, Instituto
Venezolano de Investigaciones Científicas, Caracas 1020-A,
Venezuela
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INTRODUCTION |
IN THE MAMMALIAN COLON, the
K+ secretory process implicates
several transport mechanisms at the luminal and basolateral plasma membrane of the epithelial cell (1, 11). The
Na+-K+
pump and the
Na+-K+-2Cl
cotransporter actively transport
K+ at the basolateral membrane.
This active process permits K+ to
accumulate into the colonocyte above its electrochemical equilibrium. K+ exits the cell across the
apical membrane, following its electrochemical gradient, through
Ba2+-sensitive and
tetraethylammonium (TEA)-sensitive
K+ channels. Epinephrine and the
-adrenergic agonist isoproterenol stimulate
K+ secretion (13, 19). The
enhancement of K+ secretion by
epinephrine could be due to an increase in
K+ permeability of the conductive
pathway at the apical membrane or to the stimulation of
K+ uptake across the basolateral
membrane or both.
Recently, we demonstrated that isolated colonocytes actively transport
K+ through at least three separate
mechanisms: a Na+-dependent,
ouabain-sensitive mechanism compatible with the
Na+-K+
pump; a Na+-dependent,
bumetanide-sensitive mechanism consistent with the Na+-K+-2Cl
cotransporter; and a
Na+-independent, ouabain-sensitive
mechanism identified as the colonic K+-H+
pump, in addition to passive flux (4).
The aim of the present study was to evaluate the effects of several
adrenergic agonists on K+
transport mechanisms present in isolated guinea pig colonic cells, using 86Rb as a tracer, and to
identify the second messengers involved in the response to the
adrenergic drugs.
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MATERIALS AND METHODS |
Materials.
86Rb and
[3H]polyethylene
glycol 4000 were purchased from Amersham (Little Chalfont, UK).
Epinephrine, dobutamide, clonidine, salbutamol, isoproterenol,
phenylephrine, propranolol, and benextramine were obtained from
Research Biochemicals International (Natick, MA). Fura 2-AM was
purchased from Calbiochem (La Jolla, CA). All other chemicals of
analytical grade were obtained from Sigma Chemical (St. Louis, MO) or
from Merck (Rahway, NJ).
Animals.
Male guinea pigs (weight range of 300-350 g) were used. Animals
were maintained on regular laboratory diet, were allowed free access to
water, and were starved for 24 h before being killed.
Cell isolation methods.
Colonocytes were isolated by the procedure described by del Castillo
and Sepúlveda (5). Briefly, the colon was excised from the
colonic flexure to 3 cm above the anal orifice and then rinsed, filled,
and incubated for 10 min at 37°C with solution 1 (in mM: 7 K2SO4,
44 K2HPO4,
9 NaHCO3, 10 HEPES-Tris, and 180 glucose, pH 7.4 and 340 mosmol/l). The luminal content was discarded, and the intestinal segment was refilled and incubated for 3 min at
37°C with solution 2, which
contained 0.5 mM dithiothreitol and 0.25 mM EDTA in addition to the
components of solution 1. The colon
was then gently palpated, and the luminal solution, containing isolated
cells, was collected in DMEM (100 ml) at 4°C, filtered through a
nylon mesh (60 µm pore diameter), and centrifuged at 100 g for 5 min. This step was repeated
six times to obtain six different fractions. Cells isolated during the
first two palpations (fractions
1-2) were considered surface cells, and those
isolated from the fifth and sixth palpation (fractions
5-6) were considered crypt cells. This was
confirmed in experiments that assessed the incorporation of
intraperitoneally injected
[3H]thymidine and
bromodeoxyuridine, which give both spatial and quantitative information
about cell proliferation (2, 5, 17). The isolated cells were
resuspended in DMEM and stored at 4°C in plastic tubes without
agitation. DMEM was modified to contain (in mM) 116 NaCl, 5 KCl, 1 MgSO4, 1 NaH2PO4,
7.5 K2SO4, and 10 HEPES-Tris, pH 7.4 and 320 mosmol/l. All solutions were oxygenated with 100% O2 for 15 min before use. After isolation, cells were washed three times by
centrifugation with the respective influx or efflux medium and
resuspended in the same solution. For analysis, cells were separated
from the incubation medium by centrifugation through an oil layer as
previously described (3, 4).
The cell viability was evaluated by trypan blue exclusion, oxygen
consumption, and intracellular ion concentration as previously described (3). Cells were used only when viability was >95%.
Transport experiments.
For influx experiments, cells were preincubated in DMEM for 30 min at
25°C. Uptake was initiated by adding 300 µl of preincubated cells
to 30 µl of incubation medium containing the isotopic tracer. One
250-µl sample was obtained at a time, diluted in 800 µl of incubation medium at 4°C, and immediately centrifuged at 13,000 g for 20 s. During time course
experiments, cell and incubation volumes were adjusted to obtain the
desired sample numbers, maintaining the relationship indicated above.
The intracellular radioactivity was determined after correction for the
radioactivity present in the trapped volume, measured using
[3H]polyethylene
glycol as an extracellular space marker. Control experiments showed
that fluxes measured with 86Rb, as
a tracer, were equivalents to those determined with
42K.
86Rb influx was linear for at
least 2 min at 25°C under all evaluated conditions. Influx was
measured after a 1-min incubation. As shown before (4), isolated
colonocytes transport K+ by at
least three different mechanisms: a
Na+-dependent, ouabain-sensitive
mechanism compatible with the
Na+-K+
pump; a Na+-dependent,
bumetanide-sensitive system consonant with the
Na+-K+-2Cl
cotransporter; and a
Na+-independent, ouabain-sensitive
mechanism consistent with
K+-H+
pump, in addition to a passive residual flux. To identify
K+ transport mechanisms present in
isolated colonic surface and crypt cells separately,
86Rb was measured in
Na+-containing and
Na+-free media (where
Na+ was substituted by
N-methyl-D-glucamine),
and the effects of 50 µM bumetanide and 1 mM ouabain were evaluated.
For efflux experiments, isolated cells were preincubated in DMEM
containing 0.5 µCi/ml of 86Rb
for 60 min at 25°C. Then, cells were centrifuged at 100 g for 5 min, the supernatant was
removed, and the cellular pellet was resuspended in a modified DMEM
without 86Rb (and without
K+), containing 1 mM ouabain and
50 µM bumetanide. The intracellular radioactivity was determined at
different times, and the results were expressed as a percentage of the
intracellular content at the initial time.
Intracellular
Ca2+ measuring.
Isolated crypt cells were loaded with 2 µM fura 2-AM during 30 min at
room temperature. Loaded colonocytes were stuck over a glass coverslip
with 0.1% polylysine mounted on a perfusion chamber and observed under
a microscope at ×1,000. Intracellular Ca2+ concentrations were obtained
with an SLS-100 IonOptix StepperSwitch dual-excitation light for fura
2, using an ICD-1000 intensified, charge-coupled device camera coupled
to an IonOptix fluorescence image acquisition and analysis software.
Images were acquired at 0.5 s at 340 and 380 nm. Intracellular
Ca2+ was calculated by using a
fluorescence ratio at 340:380 nm and an affinity constant
(Kd) for the
fura 2-to-Ca2+ ratio of 225 nM.
Protein determination.
Cellular proteins were determined by a modified Coomassie blue method
(10).
Statistics.
Results are expressed as means ± SE. Differences between means were
evaluated by ANOVA and considered significant at
P < 0.05. Adjustment of experimental
values to functions was made by nonlinear regression
(Marquandt-Levenberg algorithm) using a commercial program (Origen 5.0, Microcal Software, Northampton, MA).
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RESULTS |
K+ transport
mechanisms in colonic surface and crypt cells.
We have shown that isolated colonocytes transport
K+ by at least three different
mechanisms: a Na+-dependent,
ouabain-sensitive mechanism consistent with the
Na+-K+
pump; a Na+-dependent,
bumetanide-sensitive mechanism compatible with the Na+-K+-2Cl
cotransporter; and a
Na+-independent, ouabain-sensitive
mechanism identified as the
K+-H+
pump, in addition to a passive residual flux (4). The cell preparation
used in those previous studies included surface and crypt cells
together. In the present report, we separated surface and crypt cells,
following the procedure described by del Castillo and Sepúlveda
(5), and examined the K+ transport
mechanisms present in each cell type. As shown in Fig. 1, surface cells transport
K+ by two different mechanisms: a
Na+-dependent, ouabain-sensitive
mechanism consistent with the
Na+-K+
pump and a Na+-independent,
ouabain-sensitive mechanism compatible with the K+-H+
pump, in addition to passive influx. In contrast, crypt cells transport
K+ by the
Na+-K+
pump and a Na+-dependent,
bumetanide-sensitive mechanism identified as the
Na+-K+-2Cl
cotransporter, in addition to passive influx. Thus, as reported before
(5), we were unable to demonstrate
K+ transport mediated by the
Na+-K+-2Cl
cotransporter in surface cells. On the other hand, we could not identify
K+-H+
pump activity in crypt cells.

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Fig. 1.
86Rb
(K+) influx in isolated surface
(A) and crypt
(B) cells from guinea pig distal
colon. Surface and crypt cells were isolated as indicated in
MATERIALS AND METHODS.
Insets:
K+ transport mechanisms identified
in isolated cells: Na+-dependent,
ouabain-sensitive influx was assumed to be mediated by the
Na+-K+
pump; Na+-independent, ouabain-sensitive
K+ influx was considered to be
mediated by the
K+-H+
pump; Na+-dependent,
bumetanide-sensitive K+ influx was
presumed to be mediated by the
Na+-K+-2Cl
cotransporter; and the ouabain-insensitive, bumetanide-insensitive
uptake was defined as residual influx. Values are means ± SE of 3 different experiments. ** P < 0.01; *** P < 0.001; ns, not
significant, compared with controls.
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Effects of epinephrine on
K+ transport
mechanisms.
In the colon, K+ secretion is
supported by K+ uptake at the
basolateral plasma membrane of the colonic epithelial cell mediated by
the
Na+-K+-2Cl
cotransporter and the
Na+-K+
pump and K+ exit across the
luminal plasma membrane through TEA-sensitive and
Ba2+-sensitive
K+ channels (1, 11). In guinea pig
distal colon, serosal bumetanide or ouabain (13, 20) inhibits
epinephrine-stimulated K+
secretion. In the present study, we attempted to identify the K+ transport mechanisms involved
in the stimulatory response to epinephrine.
Figure 2 shows the effect of epinephrine (1 µM) on K+ transport mechanism
identified in isolated crypt cells from the guinea pig distal colon.
K+ influx was measured using
86Rb as a tracer. Epinephrine
stimulated the Na+-dependent
K+ transport mechanisms [the
Na+-K+
pump (1.5-fold) and the
Na+-K+-2Cl
cotransporter (3-fold)] and did not modify the residual influx. Epinephrine did not alter K+
transport mechanisms identified in surface epithelial cells even under
adrenergic stimulation (data not shown).

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Fig. 2.
Effect of epinephrine on K+
transport mechanisms in isolated crypt cells from guinea pig distal
colon. Cells were preincubated 15 min at room temperature with 1 µM
epinephrine, and then K+ transport
mechanisms were determined as indicated in MATERIALS
AND METHODS and Fig. 1. Values are means ± SE of 3 different experiments. ** P < 0.01; *** P < 0.001; ns, not
significant, compared with controls.
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To discriminate if the primary effect of the epinephrine was on the
Na+-K+
pump or the
Na+-K+-2Cl
cotransporter, we preincubated isolated crypt cells with epinephrine and bumetanide for 15 min and then the
K+ influx was determined. Results
are presented in Fig. 3. Preincubation of
the isolated cells with 50 µM bumetanide abolished the stimulatory effect of epinephrine on the activity of the
Na+-K+
pump, suggesting that the increase in the activity of the
Na+-K+
pump is secondary to the activation of the
Na+-K+-2Cl
cotransporter.

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Fig. 3.
Effect of epinephrine on K+
transport mechanisms in isolated crypt cells preincubated with 50 µM
bumetanide. Colonocytes were preincubated with 50 µM bumetanide and 1 µM epinephrine for 15 min at room temperature, and then
K+ influx was determined as
indicated in MATERIALS AND METHODS.
Values are means ± SE of 3 different experiments. ns, Not
significant, compared with controls.
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Figure 4 illustrates the concentration
dependence for the epinephrine effect on the bumetanide-sensitive
K+ influx in isolated crypt cells
from guinea pig distal colon. The stimulatory effect on the
Na+-K+-2Cl
cotransporter depended on the epinephrine concentration in the incubation medium, reaching the maximal effect at 1 µM with an EC50 of 91.6 ± 9.98 nM.

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Fig. 4.
Concentration dependence for the epinephrine effect on
bumetanide-sensitive K+ influx in
isolated colonic crypt cells. Points show experimental data and
represent means ± SE of 4 different experiments. Line is the best
fit to the following equation representing a sigmoidal curve: f = (a d)/[1 + (x/c)b + d], where
a = asymptotic maximum,
b = slope,
c = inflexion, and
d = asymptotic minimum.
EC50 is the value at inflection
point and represents epinephrine concentration that produced 50% of
the maximal stimulation. 2 = 11.62; r2 = 0.9997.
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Effect of different adrenergic drugs on the bumetanide-sensitive
K+ influx.
The effect of different adrenergic agonists and antagonists on the
Na+-K+-2Cl
cotransporter was examined at concentrations of
10
6 M or
10
5 M. Figure
5A shows
the effect of different adrenergic agonists on the cotransporter.
Phenylephrine (
1-agonist) and
clonidine (
2-agonist) did not
affect the bumetanide-sensitive K+
influx. However, isoproterenol
(
1- and
2-agonist), dobutamine (
1-agonist), and salbutamol
(
2-agonist) stimulated, as
epinephrine did, the activity of the
Na+-K+-2Cl
cotransporter, suggesting that the effect of the epinephrine on this
transport mechanism be mediated by
1- and
2-receptors. Figure
5B presents the effects of adrenergic
antagonists on the bumetanide-sensitive
K+ influx in isolated colonic
cells. Cells were initially preincubated for 10 min with the antagonist
and then another 15 min with epinephrine. K+ influx was evaluated as
indicated in MATERIALS AND METHODS.
Propranolol (10 µM), a
-antagonist, inhibited the stimulatory
effect of epinephrine on the bumetanide-sensitive
K+ influx, whereas benextramine
(10 µM), an
-antagonist, did not affect the cotransporter.

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Fig. 5.
Effect of different adrenergic drugs on bumetanide-sensitive
K+ influx in isolated colonic
crypt cells. Colonocytes were preincubated with the adrenergic drugs
for 15 min at room temperature, and then
K+ influx was determined as
indicated in MATERIALS AND METHODS.
Antagonists were added to preincubation medium 10 min before
epinephrine. Adrenergic agonists (A)
were used at 1 µM and adrenergic antagonists
(B) at 10 µM final concentration.
Values are means ± SE of 3 different experiments.
*** P < 0.001; ns, not
significant, compared with controls.
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Figure 6 shows a dose-response curve to
dobutamine and salbutamol on bumetanide-sensitive
K+
(86Rb) influx.
EC50 for salbutamol was six times
lower than that obtained for dobutamine, suggesting that the
-stimulatory effect is mainly mediated by
2-receptors.

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Fig. 6.
Effect of dobutamine and salbutamol on bumetanide-sensitive
K+ influx. Crypt cells were
preincubated with the agonists for 15 min at 25°C. Data are means ± SE of 3 different experiments. Points represent experimental
values and lines the best fit to a sigmoidal curve, as described in
Fig. 4.
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Concentration dependence for the propranolol effect on
epinephrine-stimulated, bumetanide-sensitive
K+ influx is shown in Fig.
7A.
Maximal effect was obtained at 10 µM propranolol with an
IC50 of 134 ± 28.2 nM. In
addition, bumetanide-sensitive K+
influx was evaluated at different epinephrine concentrations in the
presence of increasing propranolol concentrations (Fig. 7B). Experimental data were fitted
to a competitive inhibitory equation:
JK = (JK max · S)/[Kd(1 + I/Ki) + S], where
JK is
K+ influx,
JK max is
maximal K+ influx, S is
epinephrine concentration,
Kd is the
affinity constant for epinephrine, I is propranolol concentration, and
Ki is the inhibitory constant for propranolol. Using this model, we obtained a
JK max of
218 ± 6.1 nmol · mg
1 · min
1,
a Kd for
epinephrine of 91 ± 9 nM, and a
Ki for
propranolol of 72 ± 10 nM. These results confirm that the effect of
the epinephrine on the
Na+-K+-2Cl
cotransporter in colonic crypt cells is mediated by
-receptors.

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Fig. 7.
A: concentration dependence for the
propranolol effect on epinephrine-induced, bumetanide-sensitive
K+ influx in isolated colonic
crypt cells. Colonocytes were preincubated for 10 min with propranolol
and 15 additional min with epinephrine.
K+ influx was measured as
described in MATERIALS AND METHODS.
Points represent experimental data and are means ± SE of 6 different experiments. The line is the best fit to a sigmoidal curve.
IC50 is the value at inflection
point and represents propranolol concentration that produced 50% of
the maximal inhibition. 2 = 46.07; r2 = 0.9998. B: effect of propranolol on
bumetanide-sensitive K+ influx at
different epinephrine concentrations. Experimental data were fitted to
the following equation describing a competitive inhibition:
JK = (JK max · S)/[Kd(1 + I/Ki) + S], where
JK is
K+ influx,
JK max is
maximal K+ influx, S is
epinephrine concentration,
Kd is the
affinity constant for epinephrine, I is propranolol concentration, and
Ki is inhibitory
constant for propranolol. Results are means ± SE of 3 different
experiments. 2 = 52.67;
r2 = 0.9889.
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The stimulatory effect of epinephrine on the
Na+-K+-2Cl
cotransporter could be a direct effect on the cotransport mechanism or
the result of an increase in K+ or
Cl
exit of the cells that
indirectly stimulates K+ uptake
through the cotransporter. To evaluate this possibility, we examined
the effect of 10 mM TEA, 5 mM
Ba2+, and 0.1 mM DIDS, inhibitors
of K+ channels and
Cl
channels, on the
bumetanide-sensitive K+ influx in
colonic isolated cells. The results are presented in Fig.
8. Neither
Ba2+ nor TEA nor DIDS affected the
epinephrine-stimulated, bumetanide-sensitive K+ influx, suggesting that the
stimulatory effect of epinephrine on the
Na+-K+-2Cl
cotransporter is not an indirect effect determined by the activation of
K+ or
Cl
channels. Channel
blockers did not modify the
Na+-K+
pump activity and had only a small effect on residual influx (data not
shown). In addition, to discard the possibility that epinephrine could
be activating K+ channels
different from those inhibited by TEA or
Ba2+, we examined the effect of
epinephrine on K+ efflux, using
86Rb as a tracer. Isolated cells,
previously loaded with the isotope and preincubated in the presence of
epinephrine, were incubated in the absence of
86Rb (and
K+). Epinephrine did not modify
K+ efflux in colonic isolated
cells (Fig. 9). These results confirm that epinephrine
acts directly on the
Na+-K+-2Cl
cotransporter.

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Fig. 8.
Effect of Ba2+ (5 mM),
tetraethylammonium (TEA; 10 mM), and DIDS (0.1 mM) on
bumetanide-sensitive K+ influx in
isolated colonic crypt cells. Colonocytes were preincubated 15 min with
Ba2+, TEA, or DIDS and then with
or without 1 µM epinephrine. K+
flux mediated by the
Na+-K+-2Cl
cotransporter was measured as indicated in MATERIALS
AND METHODS and Fig. 1. Values are means ± SE of 3 different experiments. *** P < 0.001 compared with control.
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Fig. 9.
Effect of epinephrine on 86Rb (or
K+) efflux in isolated colonic
crypt cells from guinea pig distal colon. Cells were loaded with
86Rb for 60 min. During the last
15 min, epinephrine (1 µM) was added to the preincubation medium.
Control cells were preincubated and incubated without epinephrine.
Colonocytes were washed by centrifugation and incubated in the absence
of 86Rb (or
K+) and in the presence or not
of 1 µM epinephrine. Intracellular radioactivity was determined at
different intervals, and results are expressed as a percentage of the
intracellular content at the initial time. Values are means ± SE of
4 different experiments.
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Second messenger implicated in the stimulatory response to
epinephrine.
To identify the second messenger implicated in the stimulatory effect
of epinephrine on the
Na+-K+-2Cl
cotransport in isolated colonic crypt cells, we evaluated the effect of
dibutyryl cAMP (DBcAMP), Ca2+
chelants [EGTA and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM], and the
Ca2+ ionophore ionomycin on the
activity of the cotransporter. The results are shown in Fig.
10. The DBcAMP (0.5 mM) did not affect the
bumetanide-sensitive K+ influx.
Reducing extracellular Ca2+ to 30 nM, by the addition of EGTA to the incubation medium, did not modify
the stimulatory effect of epinephrine. However, preincubation of
isolated cells with 50 µM BAPTA-AM abolished the increase induced by
epinephrine on the activity of the cotransporter, suggesting that the
response to epinephrine is mediated by intracellular Ca2+ liberation. Increasing the
intracellular Ca2+ can reproduce
the stimulatory effect of epinephrine. Ionomycin, a
Ca2+ ionophore, induced a
significant increase in the activity of the
Na+-K+-2Cl
cotransporter. BAPTA-AM also produced an inhibitory effect on basal
bumetanide-sensitive influx (Fig. 10), suggesting that the basal
activity of the
Na+-K+-2Cl
cotransporter also depends on the intracellular
Ca2+ concentration. In addition,
we determined the effect of epinephrine on intracellular
Ca2+ concentrations in isolated
colonic crypt cells (Fig. 11). Epinephrine (10
6 M), added to the
perfusion medium, significantly increased intracellular Ca2+ concentration, in a process
inhibited by propranolol.

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Fig. 10.
Effect of dibutyryl cAMP (0.5 mM), EGTA (3 mM),
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA)-AM (50 µM), and ionomycin (5 µM) on
epinephrine-induced, bumetanide-sensitive
K+ influx in isolated crypt cells
from guinea pig distal colon. Cells were preincubated for 15 min
without addition (control), with 1 µM epinephrine, 0.5 mM dibutyryl
cAMP, 3 mM EGTA, or 3 mM EGTA and and 1 µM epinephrine or for 30 min
with 50 µM BAPTA-AM or 50 µM BAPTA-AM and 1 µM epinephrine.
Ionomycin (5 µM) was added to the incubation medium. Values are means ± SE of 3 different experiments.
* P < 0.05;
*** P < 0.001; ns, not
significant, compared with control.
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Fig. 11.
Effect of epinephrine on intracelullar
Ca2+ in isolated crypt cells.
Register represents a typical experiment showing the effect of 1 µM
epinephrine on intracellular Ca2+
concentration in a single isolated crypt cell, loaded with fura 2-AM as
indicated in MATERIALS AND METHODS.
Colonocytes were perfused with DMEM (control condition). Epinephrine
was added to the perfusion medium at 150 s after initiating the
register. At 250 s, perfusion medium was changed to DMEM without
epinephrine. This experiment was repeated 30 times with different cells
and preparations with similar results (intracellular
Ca2+ was 123 ± 7.9 nM in
control and 650 ± 75 nM with epinephrine,
n = 30).
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These results suggest that the epinephrine-induced increase in the
activity of the
Na+-K+-2Cl
cotransporter is mediated by intracellular
Ca2+ liberation.
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DISCUSSION |
Epinephrine stimulates active K+
secretion in mammalian distal colon as a result of an increase in
serosal-to-mucosal flux and a decrease in mucosal-to-serosal flux (13,
20). Epinephrine-induced K+
secretion is inhibited by serosal ouabain or bumetanide and by mucosal
Ba2+, suggesting the role of
basolateral
Na+-K+
pump and/or
Na+-K+-2Cl
cotransporter, in addition to luminal
K+ channels, in the secretory
process. The aim of the present study was to evaluate the effects of
several adrenergic agonists on the different
K+ transport mechanisms in
isolated guinea pig colonic cells, using 86Rb as a tracer, and to identify
the second massager implicated in the response to epinephrine.
In crypt cells, epinephrine stimulated
K+ uptake by increasing the
activity of the
Na+-K+-2Cl
cotransporter and the
Na+-K+
pump, without changes in the passive
K+ flux (Fig. 2). Preincubation of
isolated crypt cells with bumetanide abolished the stimulatory effect
of epinephrine on the
Na+-K+
pump (Fig. 3). This increase in the activity of the
Na+-K+
pump resulted in the activation of the
Na+-K+-2Cl
cotransporter.
However, if the epinephrine-stimulated increase in the
Na+-K+
pump was only due to an increased
Na+ delivery by the cotransporter,
then a larger epinephrine-stimulated increase in the pump would be
expected (assuming a 3:2 stoichiometry for
Na+ and
K+). Our results suggest a
possible change in the stoichiometry of the pump or an epinephrine
inhibition of a Na+ influx via a
bumetanide-insensitive pathway or activation of an ouabain- and
bumetanide-insensitive,
K+-independent
Na+ extrusion, as reported in the
small intestine and renal proximal tubule (18). Maximal effect of
epinephrine on the cotransporter was obtained with 1 µM epinephrine
with an EC50 of 91.6 nM (Fig. 4).
In contrast, we were unable to demonstrate
K+ transport mediated by the
Na+-K+-2Cl
cotransporter in surface cells, even under adrenergic stimulation (data
not shown).
The stimulatory effect of epinephrine on the
Na+-K+-2Cl
cotransporter
seems mediated by
1- and
2-receptors, since
isoproterenol (a
1- and
2-agonist), dobutamine (a
1-agonist), and salbutamol (a
2-agonist) had a stimulatory
effect on the cotransporter (Fig. 5A). The
2-effect seems to be
predominant, as indicated by the fact that the
EC50 for salbutamol is six times
lower than that obtained for dobutamine (Fig. 6).
-Adrenergic
agonists, like phenylephrine and clonidine, did not affect it. In
addition, propranolol (a
-antagonist), but not benextramine (an
-antagonist), inhibited the stimulatory effect of epinephrine on the
cotransporter (Fig. 5B). These
results suggest that the effect of epinephrine on the Na+-K+-2Cl
cotransporter is mediated by
1-
and
2-receptors.
Stimulation of K+ secretion by the
-adrenergic agonist has been demonstrated in the guinea pig distal
colon (13, 20) and rabbit distal colon (16, 21). In rabbit colon,
epinephrine reduced the short-circuit current
(Isc) and the
transepithelial potential and increased the total conductance
(Gt).
IC50 was 5 µM. These changes
were attributed to an increase in
K+ secretion without changes in
Cl
transport.
-Adrenergic agonists did not alter
Isc or
Gt. Isoproterenol had a similar effect to epinephrine, and propranolol blocked the change
in Isc and
Gt produced by
epinephrine. Thus the effect of epinephrine seems to be due to a direct
stimulation of colonic epithelial cells mediated by
-adrenergic
receptors (16, 21).
The inability of Ba2+, TEA, or
DIDS to inhibit epinephrine enhancement of the activity of the
cotransporter, in isolated crypt cells, indicates that this stimulatory
response is not an indirect effect mediated by the opening of
TEA-sensitive or Ba2+-sensitive
K+ channels nor DIDS-sensitive
Cl
channels (Fig. 7).
Moreover, epinephrine did not modify
K+
(86Rb) efflux in isolated crypt
cells (Fig. 8). These results support the idea that the stimulatory
effect of epinephrine on the
Na+-K+-2Cl
cotransporter is not an indirect effect induced by the opening of
K+ or
Cl
channels.
cAMP or Ca2+ mediates ion
secretion in many epithelial cells as second messengers. Usually, cAMP
has been recognized as the second messenger involved in the cellular
response to activation of
-adrenergic receptors (22). However, in
some cell systems, different second messengers (9, 14) can mediate the
response to
-adrenergic agonists. To identify the second messenger
related to the stimulatory effect of epinephrine on the
Na+-K+-2Cl
cotransporter in isolated crypt cells, we examined the effect of cAMP
and Ca2+ on the
Na+-K+-2Cl
cotransporter. As shown before (5), DBcAMP (0.5 mM) was unable to
stimulate the cotransporter. Reduction of the extracellular Ca2+ to 30 nM, by the addition of
EGTA to the incubation medium, did not significantly affect the
response to epinephrine. In contrast, using BAPTA-AM as an
intracellular Ca2+ chelator, it
was possible to abolish the stimulatory response to epinephrine in
isolated crypt cells, indicating that an increase in intracellular
Ca2+ is essential to maintain the
response to epinephrine. Furthermore, ionomycin, a
Ca2+ ionophore, also stimulated
the activity of the cotransporter. In addition, epinephrine increases
intracellular Ca2+ concentration
in isolated cells (Fig. 11). These results show that
Ca2+ serves as second messenger in
the stimulatory response to epinephrine in crypt cells.
The mechanism by which the adrenergic agonist modifies
K+ secretion in mammalian colon
has not been well defined. Both cAMP and
Ca2+ have been shown to increase
K+ secretion in colonic epithelium
(1, 6, 11). However, isoproterenol does not increase intracellular cAMP
levels in human colon (7) and
Cl
secretion is not
increased by epinephrine (20). These results suggest that the
-adrenergic stimulation of K+
secretion, as well as the increase in the activity of the
Na+-K+-2Cl
cotransporter in crypt cells, is not mediated by cAMP. On the other
hand, removal of Ca2+ of the
serosal solution partially inhibits the decrease in
Isc produced by
epinephrine (21) and the addition of A-23187 (19, 20) or ionomycin (8),
Ca2+ ionophores, induces an
increase in K+ secretion. These
findings suggest that Ca2+ could
mediate the enhancement of K+
secretion by
-agonists.
It should be noted that cAMP did not stimulate the
Na+-K+-2Cl
cotransport (Fig. 10). This observation raises the question about why
the activation of cAMP-dependent
Cl
channels did not
stimulate the
Na+-K+-2Cl
cotransporter. A possibility could be that a stimulation of
Na+-K+-2Cl
cotransport is not observed because cAMP may also activate a basolateral K+ conductance,
leading to underestimation of 86Rb
influx due to a basolateral recycling. If this is the case, cAMP must
stimulate 86Rb efflux. However,
neither 0.5 mM DBcAMP, 0.5 mM bromo-cAMP, nor 100 µM forskolin
produced any effect on 86Rb efflux
(data not shown). In addition, Deiner et al. (6), using whole cell
patch-clamp recordings, showed that forskolin, vasoactive intestinal
polypeptide, or membrane-permeable cAMP analogs decreased
K+ conductance in rat distal
colon. Furthermore, Maguire et al. (15) observed an inhibition of
basolateral K+ conductance in
nystatin-permeabilized human colon by forskolin. These reports and our
experiments suggest that cAMP did not significantly modify basolateral
K+ conductance in colonic cells. A
second possibility is that, under our experimental conditions,
Cl
flux through
Cl
channels must be low.
Intracellular Cl
concentration in isolated crypt cells was 93 ± 10.5 mM, and the addition of 20 mM K+ to the medium
induces cellular depolarization. These conditions may generate a
relatively small electrochemical gradient for
Cl
flux through
cAMP-dependent Cl
channels,
even if they are open.
Interestingly, epinephrine stimulated the cotransporter in crypt but
not in surface cells (Fig. 2). This observation agrees with that
reported by Halm and Rick (12), which indicates that the secretory
process related with K+ secretory
response to epinephrine, in guinea pig distal colon, was restricted to
the lower two-thirds of the crypt of Lieberkühn.
In summary, we have shown that the
K+ transport mechanism implicated
in the response to epinephrine, in isolated crypt cells from guinea pig
distal colon, is the
Na+-K+-2Cl
cotransporter, in a process mediated by
1- and
2-receptors and modulated by
intracellular Ca2+ liberation. Our
results suggest that the stimulatory effect of epinephrine on
K+ secretion seems primarily
mediated by the activation of K+
entry across the basolateral plasma membrane through the
Na+-K+-2Cl
cotransporter.
This study formed part of J. C. Arévalo's Magister Scientiarum
Thesis at Centro de Estudios Avanzados of the Instituto Venezolano de
Investigaciones Científicas, 1997.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. R. del
Castillo, Centro de Biofísica y Bioquímica, IVIC,
Apartado 21827, Caracas 1020-A, Venezuela (E-mail:
jdelcas{at}cbb.ivic.ve).