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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (+/?).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

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.