The primary and final effector mechanisms required for
kinin-induced epithelial chloride secretion
Alan W.
Cuthbert1 and
Clare
Huxley2
1 Department of Pharmacology,
University of Cambridge, Cambridge CB2 1QJ; and
2 Department of Biochemistry and
Molecular Genetics, St. Mary's Hospital Medical School, London W2
1PG, United Kingdom
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ABSTRACT |
The short-circuit current technique was used to
examine the effects of
N2-L-lysylbradykinin
(LBK) on chloride secretion in the mucosae of the mouse intestine. It
was found to be a potent chloride secretagogue in the mucosa lining the
colon, jejunum, and cecum, as it is in most mammals, with 2 nM being
sufficient to cause half-maximal secretion. The extent of the responses
was in the order cecum > colon > jejunum. In cystic fibrosis (CF)
null mice, with no CF transmembrane conductance regulator (CFTR)
chloride channels, LBK caused no chloride secretion, but transporting
activities for other ions were revealed. Introduction of the human CF
gene into the genome of CF null mice at the zygote stage restored the chloride secretory activity of LBK, with only minor differences in
potency. In mice in which the kinin
B2 receptor gene had been disrupted, LBK had no effect, whereas the responses to forskolin were
unchanged. Thus the acute effects of kinins on chloride secretion depend uniquely on kinin B2
receptors and CFTR chloride channels, which form the primary and final
effector mechanisms of the secretory process.
cystic fibrosis transmembrane conductance regulator; yeast
artificial chromosome; kinin B2
receptors
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INTRODUCTION |
THE PURPOSE OF THIS STUDY was to determine
unequivocally the primary and final effector mechanisms involved in
kinin-induced Cl
secretion
in the epithelium of the mammalian gut. Kinins are some of the most
powerful stimulants of Cl
secretion known and are released in pathophysiological conditions such
as secretory diarrhea (30) and infections of the airways (21). Kinins act through a cascade of adenosine
3',5'-cyclic monophosphate (cAMP), prostaglandins, and
intracellular Ca2+ as second
messengers (13), but here we define both the type of
Cl
channel in the apical
membranes that allows exit of
Cl
into the lumen and also
the nature of the receptors in the basolateral side of the mucosa with
which kinins interact. Epithelial tissues from genetically modified
mice have been used in which the genes for particular proteins involved
with the process of signal recognition (i.e., receptors) and with the
efflux of Cl
(i.e.,
channels) have been disrupted or replaced with alternate genes.
Reliance has not been placed on blocking drugs whose actions may not be
entirely specific. For example,
Cl
channel-blocking drugs,
such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid
and glibenclamide, have multiple actions at other sites (5). Two, or
possibly three, types of kinin receptors have been identified, known as
B1,
B2, and
B3 receptors (27). Certain
classical B2-receptor antagonists,
such as HOE-140, inhibit kinin actions at some
B1 receptors (22).
All of the experiments have been made with mucosae, namely those of the
colon, cecum, and jejunum, from the mouse alimentary tract. Few
investigators have explored the actions of kinins on mouse tissues, and
consequently some initial observations are made on the sensitivity and
maximal secretory activity in response to kinins. Throughout,
N2-L-lysylbradykinin
(LBK) has been used as the archetypal agonist.
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MATERIALS AND METHODS |
All experiments were made with mice maintained under approved
conditions within an animal facility and killed by an approved method,
i.e., exposure to an increasing concentration of
CO2. Mucosae from the
colon, jejunum, and cecum of mice were dissected free of associated
muscle layers and mounted in Ussing chambers (window area, 20 mm2) for voltage clamping at
zero potential. This was carried out with a World Precision Instruments
dual-voltage clamp, using series-resistance voltage compensation.
Current-passing electrodes consisted of 3 M KCl-agar tubes inserted
into the chambers as far as possible from the tissue. These electrodes
led, via Ag-AgCl electrodes, to the output stage of the amplifier. The
potential-sensing electrodes consisted of Krebs-Henseleit solution
(KHS)-filled tubes with their ends as close to the tissue as possible
and connected via calomel cells to the amplifier input stage. No more
than two pieces of mucosa of each type were taken from a single animal.
Both wild-type and heterozygote null mice were used as controls, as no
differences in the responses to secretagogues between wild-type and
heterozygote tissues are reported (7). Each epithelium was bathed on
both sides with 20 ml KHS, oxygenated by bubbling with 95%
O2-5%
CO2, and maintained at 37°C.
KHS had the following composition (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4,
25 NaHCO3, and 11.1 glucose.
Four types of mice were used for the experiments:
1) wild-type mice,
2) cystic fibrosis (CF) null mice
(Cftrtm1Cam),
3) kinin
B2 receptor null mice, and
4) YAC mice. CF null mice were
generated as follows. Exon 10 of the CF gene was disrupted using
homologous recombination techniques in embryonic stem cells, after
which they were inserted into blastocysts and implanted into surrogate
mothers. Chimeric heterozygous offspring were used to generate
homozygous null CF animals (24). To generate mice in which the kinin
B2 receptor gene was disrupted,
the same technique as for CF was used to give mice without
B2 receptors (4). YAC mice are
animals in which a yeast artificial chromosome was incorporated into
the mouse genome. Human DNA fragments including the human CF gene plus
upstream promoter elements contained in a YAC were injected into the
pronucleus of murine CF zygotes. After implantation and gestation, mice
with the CF genotype but which had incorporated the human CF gene in
the genome were obtained. Full details of this procedure are given
elsewhere (19).
The following drugs (all obtained from Sigma) were used in the study:
forskolin, furosemide, and LBK. Tests for significance were made using
an unpaired Student's t-test, with
P < 0.05 being considered
significant.

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Fig. 1.
Concentration-response curves for
N2-L-lysylbradykinin
(LBK) in wild-type colonic (A),
jejunal (B), and cecal
(C) mucosae. LBK was added to
basolateral bathing solution. After exposure to a given peptide
concentration, tissue was washed thoroughly and 90 min allowed for
recovery before the next concentration was added. Specimen responses
for single tissues of 20 mm2 are
depicted. Means ± SE are shown
(n = 6 for colon, 8 for jejunum, and 7 for cecum). Isc, short-circuit current.
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Fig. 2.
Examples of responses of cystic fibrosis (CF) null mucosae (20 mm2) to LBK.
A: colon.
B: jejunum.
C: cecum. Peptide was added to the
basolateral face of the epithelia at a concentration of 1 µM (for the
colon) and 300 nM (for the jejunum and cecum). Furosemide (1 mM) was
added after LBK in all tissues, again to the basolateral side. In some
instances, forskolin (10 µM) was added to both sides of the tissues.
At right of each trace is a comparison
of the responses of wild-type tissues with those of CF null mice when
exposed to the same supramaximally effective concentrations of LBK.
Controls were a mixture of wild-type and heterozygote tissues (+/?).
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RESULTS |
Concentration-response relationships for LBK on murine colonic,
jejunal, and cecal mucosae were determined. These relationships provide
a benchmark against which the sensitivity to kinins could be judged in
genetically modified tissues. Figure 1 shows that the
half-maximal effective concentration
(EC50) is in the low nanomolar
range (2-5 nM) and was independent of tissue type, whereas the
maximal Cl
secretory
responses were variable, being 139.1 ± 18.7 µA · cm
2
(n = 6) in the colon, 33.9 ± 4.2 µA · cm
2
(n = 8) in the jejunum, and 178.0 ± 17.0 µA · cm
2
(n = 7) in the cecum
at the highest concentrations of LBK used. Some submaximal
concentrations of LBK produced marginally greater responses than higher
concentrations, a result of rapid desensitization and receptor
internalization (29). Ninety minutes were allowed for recovery after
kinin removal before kinin was reapplied. Similar experiments were made
with mucosae from CF null mice with the previously used maximal
concentrations. Here LBK caused only reductions in short-circuit
current (Isc) that were small by comparison with the Cl
secretory responses
in wild-type tissues, examples of which are depicted in Fig.
2. In the colon and cecum, the reduction in
Isc was reversed by furosemide. In the jejunum,
LBK is able to reverse the small Isc increase
caused by forskolin and vice versa, and the LBK effect was not reversed
by furosemide. Thus epithelia from CF null mice demonstrate minor
electrogenic transporting activities caused by LBK, which are obscured
in normal epithelia by the presence of dominant
Cl
secretion. The
histograms (Fig. 2) show the cumulative data for the effects of LBK on
CF mucosae.
The next series of experiments was made with mice of the CF genotype,
but in which the human CF transmembrane conductance regulator (CFTR)
gene had been incorporated in the genome, i.e., YAC mice. Using the
same maximal concentrations of LBK, we investigated colonic, cecal, and
jejunal mucosae from YAC mice. The data are given in Fig.
3. All the YAC epithelia responded to LBK
with an increase in Isc that was sensitive to
furosemide, in contrast to the CF null epithelia, even though they came
from mice with the CF null genotype. The responses in the colon and
jejunum were not significantly different from those in wild-type
tissues, whereas those in the cecum were significantly less
(P < 0.05). Concentration-response curves were also determined for YAC epithelia; the
EC50 was ~2 nM in the cecum and
jejunum but was somewhat higher (25 nM) in the colon (Fig.
4).

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Fig. 3.
Comparison of responses to LBK in mucosae from yeast artificial
chromosome (YAC) mice (open bars) to those from wild-type animals
(solid bars). LBK was applied to basolateral faces of tissues (1.0 µM
in the colon and 300 nM in the cecum and jejunum). Responses in ceca
from YAC mice were significantly less than for wild-type animals
(P < 0.05).
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Fig. 4.
Concentration-response curves for LBK in YAC mice colonic
(A), jejunal
(B), and cecal
(C) mucosae. Procedures were as
described for Fig. 1. Means ± SE are shown;
n = 4 throughout.
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B2 receptor null mice, in which
the B2 receptor gene had been
disrupted, were used to examine whether epithelial
Cl
secretion was
exclusively dependent on this type of receptor. With the use of maximal
LBK concentrations, no responses were observed in the three types of
intestinal epithelia, yet responses to forskolin were comparable to
those seen in wild-type epithelia (Figs. 5
and 6).

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Fig. 5.
Examples of responses to LBK in a colonic, jejunal, and cecal
epithelium from a single B2
receptor null mouse. LBK was applied basolaterally (1.0 µM for colon
and 300 nM for jejunum and cecum) followed by forskolin (10 µM) and
furosemide (1 mM). Each epithelium had an area of 20 mm2.
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