Mechanisms of Chloride and Bicarbonate Secretion Across Rat Proximal Colon Evoked by a CO Donor

Abstract

The aim of the present study was to investigate whether carbon monoxide induces changes in ion transport across rat proximal colon. In Ussing chamber experiments, tricarbonyldichlororuthenium(II) dimer (RuCO), a CO-donor, evoked a concentration-dependent increase in short-circuit current (I_sc). Anion substitution experiments suggest that a secretion of Cl⁻ and HCO₃ ⁻ underlie the RuCO-induced current. Partial inhibition by the neurotoxin tetrodotoxin suggests the involvement of secretomotor neurons in this response. Consequently, endogeneous carbon monoxide might be a physiological modulator of colonic ion transport.

Key words: carbon monoxide, CFTR, Cl⁻/HCO₃ ⁻ secretion, proximal rat colon

Introduction

Recently it has been shown that small gaseous molecules are able to regulate epithelial functions. The first gasotransmitter, which has been described, was nitric oxide (NO) [Waldmann and Murad 1987]. Increased production of nitric oxide in the colon results in a strong Cl⁻ secretion, caused by the opening of apical Cl⁻ channels and basolateral K⁺ channels as well as stimulation of the basolateral Na⁺-K⁺-pump [8].

In contrast, carbon monoxide (CO) is often considered only as a respiratory toxin. However, apparently carbon monoxide is a gasotransmitter involved in the regulation of several cell functions, too. For example, CO has strong actions on blood circulation as it induces a vasodilation [5]. Carbon monoxide is produced during the degradation of hemoglobin. This process is catalysed by two enzymes, the inducible isoform hemoxygenase I and the constitutive isoform hemoxygenase II [6].

Carbon monoxide as a small gaseous molecule is able to cross membranes via diffusion. Obviously, CO (like NO) can stimulate the soluble guanylate cyclase and thereby stimulate the intracellular production of cGMP [3]. Furthermore, CO is able to activate K⁺ channels directly, leading to a hyperpolarisation of the membrane [5]. In H441 cells, i.e. human bronchiolar epithelial cells, CO inhibits Na⁺ channels independent from cGMP; an effect, which may be caused by a modification of histidine residues in the ion channels (or regulators of them) as concluded from experiments with diethyl pyrocarbonate, a histidine-modifying agent [1].

Enteric neurons, the key players in the regulation of intestinal transport, express the enzymes for CO production [2, 7]. So carbon monoxide apparently acts as a gasotransmitter in the enteric nervous system which is able to regulate gastrointestinal functions largely independent from the central nervous system [11]. In the colonic tumour cell line, Caco-2, a CO-donor or pretreatment with hem to stimulate endogenous CO production causes Cl⁻ secretion [9]. There is, however, no information available about the effects of carbon monoxide on the native intestinal epithelium. Therefore the aim of the present study was to investigate whether CO induces changes in ion transport across rat proximal colon and to study the mechanisms involved.

Material and methods

Ussing chamber experiments

For Ussing chamber experiments, femal and male Wistar rats (200–250 g) were used. The experiments were carried out in a bathing solution containing (mmol·l⁻¹): 107 NaCl, 4.5 KCl, 25 NaHCO₃, 1.8 Na₂ HPO₄, 0.2 NaH₂ PO₄, 1.25 CaCl₂, 1 MgSO₄, 12.2 glucose (gassed with 5% (vol/vol) CO₂/95% (vol/vol) O₂; pH 7.4). For the Cl⁻-free buffer, NaCl and KCl were replaced equimolarly by Na gluconate and K gluconate. When in addition HCO₃ ⁻ and Cl⁻ were omitted, a HEPES (N-(2-hydroxyethyl)piperazine-Nʹ-2-ethansulfonic acid)-buffered Tyrode solution with (mmol·l⁻¹): 140 Na gluconate, 5.4 K gluconate, 10 Ca gluconate, 1 MgSO₄, 10 HEPES, 12.2 glucose (gassed with O₂; pH of 7.4) was used. Ussing chamber experiments with mucosa-submucosa preparations of rat proximal colon were performed as previously described [8].

Statistics

Results are given as mean ± standard error of the mean (SEM) with the number (n) of investigated tissues. For the comparison of two groups, either a Student's t-test or a Mann Whitney U-test was applied. An F-test decided which test method had to be used.

Results

RuCO evokes chloride secretion across rat proximal colon

Tricarbonyldichlororuthenium(II) dimer (RuCO) evoked a concentration-dependent increase in short-circuit current (I_sc) (Fig. 15.1). A maximal response was achieved with a concentration of 2.5 · 10⁻⁴ mol·l⁻¹. A first significant increase in I_sc was evoked at a concentration of 10⁻⁵ mol·l⁻¹ at the serosal and 5·10⁻⁵ mol·l⁻¹ at the mucosal side of the tissue. Repeated administration of RuCO resulted in a desensitization. The second administration of RuCO (2.5·10⁻⁴ mol·l⁻¹ at the serosal side) caused an increase in I_sc of only 45.1 ± 18.2% (n = 7) in comparison to the first administration, and a third administration of only 38.2 ± 8.3% (n = 7). Tissue conductance (Gt) after RuCO application (2.5·10⁻⁴ mol·l⁻¹ at the serosal side) increased by 6.2 ± 2.7 mS·cm⁻² (n = 7).

Fig. 15.1 Concentration-dependent increase in I_sc across rat proximal colon evoked by tricarbonyldichlororuthenium(II) dimer (RuCO) administered either at the serosal (squares) or the mucosal (circles) side. Data are given as increase in I_sc above baseline (Δ I_sc) just prior administration of the CO donor and are means (symbols) ± SEM (lines); n = 7.

Ionic nature of the I_sc response

Pretreatment of the tissue with glibenclamide (5·10⁻⁴ mol·l⁻¹ at the mucosal side), an inhibitor of the CFTR channel [4], resulted in a reduced RuCO effect (Table 15.1). In addition, anion substitution experiments were conducted. Substitution of Cl⁻ significantly inhibited the I_sc induced by RuCO. After replacement of HCO₃ ⁻ and Cl⁻, the increase in I_sc evoked by RuCO was nearly abolished (Table 15.1).

Table 15.1 RuCO effect across rat proximal colon.

Drug tested	Δ Isc with drug/anion substitution (µEq·h−1·cm−2)	Δ Isc without drug (µEq·h−1·cm −2)
Anionic dependency and sensitivity against transport inhibitors of the Isc response evoked by RuCO (2.5 · 10 −4 mol·l −1 at the serosal side). For each experimental series, the response to RuCO was tested after anion substitution or in the presence of a transport inhibitor (middle column), and compared with the response under control conditions (right column). Inhibitors used were: glibenclamide (5 · 10 −4 mol·l −1 at the mucosal side) or tetrodotoxin (TTX; 10 −6 mol · l −1 at the serosal side). Data are given as difference against the baseline just prior administration of RuCO (Δ Isc) and are means ± SEM, n = 6–9, * P < 0.05 versus response to RuCO in the absence of the respective drug.
Substitution of Cl−	0.121 ± 0.035*	1.009 ± 0.311
Substitution of Cl− and HCO3 −	0.044 ± 0.025*	1.219 ± 0.374
Glibenclamide	0.250 ± 0.119	0.803 ± 0.211
Tetrodotoxin	0.321 ± 0.088	0.763 ± 0.166

Involvement of secretomotor neurons in the RuCO effect

To investigate a possible involvement of secretomotor neurons in the RuCO-induced I_sc, these neurons were inhibited with the neurotoxin, tetrodotoxin (TTX). In the presence of this neurotoxin (10⁻⁶ mol·l⁻¹ at the serosal side), the RuCO-induced I_sc was reduced (Table 15.1), although this inhibition did not reach statistical significance.

Discussion

The present results indicate that the CO donor, RuCO, is able to induce anion secretion across rat proximal colon. Both Cl⁻ and HCO₃ ⁻ had to be removed from the bathing solution, before the I_sc response evoked by RuCO was abolished (Table 15.1). This is, however, not unexpected, because the CFTR anion channel, i.e. the dominant anion channel in the apical membrane, is permeable both for Cl⁻ as well as for HCO₃ ⁻ [4].

Consequently, exogenous CO has the ability to modify ion transport across the epithelium of the proximal colon. This rises the question, whether CO might act as modulator of epithelial functions under physiological conditions. Enteric neurons, which regulate nearly all functions of the gastrointestinal tract, express the enzymes for CO production. For example, hemoxygenase II is found in the human myenteric plexus [7] as well as in the myenteric and submucosal plexus of rat ileum [2], so neuronally released CO might well effect epithelial transport processes. Interestingly, the partial sensitivity of the RuCO-induced I_sc suggests that enteric neurons might not only be producers of this gasotransmitters but might in addition act as targets for this emerging new transmitter.

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Correspondence
J. Steidle
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