Segment-specific effects of epinephrine on ion transport in the colon of the rat

Silke Hörger, Gerhard Schultheiß, and Martin Diener

Institut für Veterinär-Physiologie, Universität Giessen, D-35392 Giessen, Germany

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The effect of epinephrine on transport of K+, Na+, Cl-, and HCO-3 across the rat colon was studied using the Ussing chamber technique. Epinephrine (5 × 10-6 mol/l) induced a biphasic change in short-circuit current (Isc) in distal and proximal colon: a transient increase followed by a long-lasting decay. The first phase of the Isc response was abolished in Cl--poor solution or after bumetanide administration, indicating a transient induction of Cl- secretion. The second phase of the response to epinephrine was suppressed by apical administration of the K+ channel blocker, quinine, and was concomitant with an increase in serosal-to-mucosal Rb+ flux, indicating that epinephrine induced K+ secretion, although this response was much smaller than the change in Isc. In addition, the distal colon displayed a decrease in mucosal-to-serosal and serosal-to-mucosal Cl- fluxes when treated with epinephrine. In the distal colon, indomethacin abolished the first phase of the epinephrine effect, whereas the second phase was suppressed by TTX. In the proximal colon, indomethacin and TTX were ineffective. The neuronally mediated response to epinephrine in the distal colon was suppressed by the nonselective beta -receptor blocker, propranolol, and by the beta 2-selective blocker, ICI-118551, whereas the epithelial response in the proximal colon was suppressed by the nonselective alpha -blocker, phentolamine, and by the selective alpha 2-blocker, yohimbine. These results indicate a segment-specific action of epinephrine on ion transport: a direct stimulatory action on epithelial alpha 2-receptors in the proximal colon and an indirect action on secretomotoneurons via beta 2-receptors in the distal colon.

enteric nervous system; chloride transport; potassium transport; prostaglandins

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE EPITHELIUM OF THE COLON is able to both absorb and secrete K+. To absorb K+, K+ enters the colonocyte from the colonic lumen via an H+-K+-ATPase; K+ probably leaves the cell by passing through basolateral K+ channels. K+ secretion is driven by the intracellular accumulation of K+ via the basolateral Na+-K+-2Cl- cotransporter and the Na+-K+-ATPase; the exit into the colonic lumen is mediated by apical K+ channels (for review, see Ref. 1).

These transport pathways are under the extracellular control of neurotransmitters, hormones, and paracrine substances. One of these extracellular messengers affecting intestinal K+ transport is epinephrine. This adrenergic agonist has been shown to induce a K+ secretion in rabbit (12, 22) and guinea pig colon (18), which leads to a decrease in short-circuit current (Isc). Also, in the rat colon, epinephrine has been shown to induce a decrease in Isc (17). In addition to the induction of K+ secretion, in guinea pig colon, epinephrine has been observed to transiently stimulate Cl- secretion (18). In other intestinal segments such as rabbit ileum (10), rat colon (17), or rabbit proximal colon (21) epinephrine has been reported to stimulate Na+ and Cl- absorption and/or to inhibit Cl- secretion, whereas in rabbit distal colon no changes in Na+ and Cl- transport were evoked (22). No data are available concerning the effect of epinephrine on K+ transport across the rat colon.

Therefore, the aim of the present experiments was to study the action of epinephrine on the transport of K+, Na+, Cl-, and HCO-3 across the rat colon using electrophysiological and pharmacological approaches. Because of the known segmental differences across the longitudinal axis of the large intestine (see, e.g., Ref. 15), the experiments were performed with the distal and the proximal colon.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Solutions. For the Ussing chamber experiments, a Parsons solution was used containing (in mmol/l) 107 NaCl, 4.5 KCl, 25 NaHCO3, 1.8 Na2HPO4, 0.2 NaH2PO4, 1.25 CaCl2, 1 MgSO4, and 12 glucose. The solution was gassed with carbogen (5% CO2 in 95% O2); pH was 7.4. When 86Rb+ fluxes were measured, KCl was replaced by RbCl. For the Cl--poor buffer, NaCl was replaced with sodium gluconate (elevating the Ca2+ concentration to 5.75 mmol/l to compensate for the Ca2+-buffering properties of gluconate). For the indirect measurement of HCO-3 transport, an unbuffered Parsons solution was used consisting of (in mmol/l) 135.8 NaCl, 4.5 KCl, 1.25 CaCl2, 1 MgSO4, and 12 glucose. This solution was gassed with O2 and was prepared from degassed distilled water.

Tissue preparation. Rats of both sexes were used (weight of 180-220 g). The animals had free access to water and food until the day of the experiment. Animals were stunned by a blow on the head and killed by exsanguination (approved by Regierungspräsidium Giessen, Giessen, Germany). The appearance of palmlike foldings distinguished between the border and the distal and proximal colon (14). The serosa and muscularis propria were stripped away by hand to obtain a mucosa-submucosa preparation.

Isc measurement. The mucosa-submucosa preparation was fixed in a modified Ussing chamber, bathed with a volume of 3.5 ml on each side of the mucosa (9). The tissue was incubated at 37°C and short-circuited by a voltage clamp (Ing. Büro für Mess- und Datentechnik, Dipl. Ing. K. Mussler, Aachen, Germany) with correction for solution resistance. Isc was continuously recorded every 6 s; tissue conductance (Gt) was measured every minute. To compare different experimental periods during measurement of unidirectional ion fluxes, the means of the electrical parameters (measured every 5 min) over the whole period were calculated.

Experimental design. In some experiments, i.e., the concentration response and the desensitization experiments, epinephrine was administered repeatedly to the same tissue. In this case, the serosal compartment was washed three times in 5-min intervals with five times the chamber volume before the drug was administered a second or third time. In all other experiments, epinephrine was administered only once to the same preparation. The maximal increase (first phase) and the maximal decrease in Isc (second phase) evoked by epinephrine were measured in the presence and absence (control) of putative inhibitors. Inhibitors were administered to the compartment indicated in the text; Isc was allowed to stabilize, which usually took 15-20 min, before epinephrine was added. In some experiments, the half time of the epinephrine response was calculated by linear interpolation of the change in Isc.

Unidirectional flux measurements. After an equilibration period of 60 min, 22Na (59 kBq) and 36Cl (29 kBq) were added to one side of the epithelium (9). After an additional 20 min to allow isotope fluxes to reach a steady state, unidirectional ion fluxes were determined in two sequential 20-min periods. The protocol was as follows: first phase, control; second phase, fluxes 5 min after administration of epinephrine (5 × 10-6 mol/l at the serosal side). From the measured unidirectional fluxes, net ion fluxes (Jnet) were calculated. Residual ion flux (Rnet), i.e., the sum of the movement of all ions other than Na+ and Cl-, was calculated according to the following: Rnet Isc - Nanet + Clnet. A positive Rnet indicates either the absorption of a cation or the secretion of an anion. With the same protocol, the effect of epinephrine on unidirectional Rb+ fluxes was determined. The amount of 86Rb+ added into the chambers was 37 kBq.

Serosal-to-mucosal HCO-3 flux was estimated from the base transport into the mucosal compartment (24). The serosal half chamber was filled with standard Parsons solution gassed with CO2-O2, whereas the fluid in the mucosal compartment was an unbuffered Parsons solution (see Solutions) gassed with O2, from which samples were taken and replaced by fresh solution in regular intervals. The base transport was measured in four sequential 30-min periods, i.e., two control periods and two periods in the absence (control) or presence of epinephrine (5 × 10-6 mol/l at the serosal side).

The volume of HCl (0.5 mmol/l) needed to titrate the mucosal sample to a pH of 7.0 was measured; from this value, the amount of HCO-3 in the sample was calculated using a calibration curve with samples of known HCO-3 concentration. The calibration curve was linear (r > 0.996) over the complete concentration range tested (0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mmol/l).

Chemicals. Methazolamide and phentolamine methanesulfate (Aldrich, Steinheim, Germany) were dissolved in DMSO (final concentration 0.3%, vol/vol). Bumetanide, indomethacin, quinine hydrochloride, and yohimbine hydrochloride were dissolved in ethanol (final maximal concentration 0.1%, vol/vol). Atenolol (gift from Zeneca, Plankstadt, Germany), atropine sulfate, epinephrine bitartrate, hexamethonium chloride, ICI-118551 {1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride; Tocris, Bristol, UK}, prazosin hydrochloride (gift from Pfizer, Karlsruhe, Germany), and propranolol (Aldrich) were dissolved in aqueous stock solutions diluted in salt buffer just before use. TTX was dissolved as a stock solution in citrate buffer (20 mmol/l). Tetraethylammonium (TEA) was added as Cl- salt. If not indicated differently, drugs were from Sigma (Deisenhofen, Germany). Radiochemicals were obtained from NEN Life Science (Köln, Germany). Specific activity was 3.9 TBq/g Na+ and 550 MBq/g Cl-; the initial activity of 86Rb+ amounted to 411 GBq/g.

Statistics. Values are given as means ± SE. Significances were tested by paired or unpaired two-tailed Student's t-test or a U test, respectively. An F test decided which test method was to be used; P < 0.05 was considered to be statistically significant. Statistical comparison of qualitative data, i.e., the absence or the presence of a first or a second phase of the epinephrine response, was tested by a chi 2 test. The quality of linear regressions was checked by the linear regression coefficient (r).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effect of epinephrine: basic properties. After an equilibration time of 1 h, the distal colon exhibited a spontaneous baseline Isc of 3.4 ± 0.1 µeq · h-1 · cm-2 (n = 86) and a Gt of 15.7 ± 0.8 mS/cm2 (n = 86). In the proximal colon, baseline Isc amounted to 3.4 ± 0.1 µeq · h-1 · cm-2 (n = 85) at a Gt of 32.4 ± 2.3 mS/cm2 (n = 85, P < 0.05 vs. distal colon). Epinephrine (5 × 10-6 mol/l at the serosal side) induced a biphasic change in Isc in both colonic segments (Fig. 1). A first transient increase in Isc, which amounted to 0.2 ± 0.0 µeq · h-1 · cm-2 in the distal (n = 86, P < 0.05) and 0.3 ± 0.0 µeq · h-1 · cm-2 in the proximal colon (n = 85, P < 0.05), was followed by a long-lasting decrease in Isc of 0.9 ± 0.1 µeq · h-1 · cm-2 in the distal (n = 86, P < 0.05) and of 0.7 ± 0.1 µeq · h-1 · cm-2 in the proximal colon (n = 85, P < 0.05; Table 1). The first phase of the effect of epinephrine developed rapidly with a half time of about 30 s, whereas the half time of the second phase amounted to 140-160 s (Table 1). The second phase of the epinephrine effect was accompanied by an increase in Gt, which was about nine times greater in the proximal compared with the distal colon (P < 0.05; Table 1).


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Fig. 1.   Effect of epinephrine (5 × 10-6 mol/l at the serosal side) on short-circuit current (Isc) in the distal (A) and proximal (B) rat colon. Tracings are representative for 85-86 experiments with each tissue; for statistics, see Table 1.

                              
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Table 1.   Effect of epinephrine on electrical parameters and half time of the Isc response

The first phase of the epinephrine response exhibited a more pronounced variability compared with the second phase. When the presence of each individual phase was defined as change of at least 0.1 µeq · h-1 · cm-2, in the distal colon only 60 of 86 tissues (70%) showed a first phase, whereas 80 of 86 tissues (93%) exhibited a second phase of the epinephrine response (P < 0.05 vs. first phase, chi 2 test). In the proximal colon, the numbers were 79% (67 of 85) for the first and 92% (78 of 85) for the second phase (P < 0.05 vs. first phase, chi 2 test).

The effect of epinephrine, especially the second phase, i.e., the decrease in Isc induced by the drug, showed a relative flat concentration dependence (Fig. 2). However, a clear first phase, i.e., an increase in Isc, was only observed when the highest concentration (5 × 10-6 mol/l) was administered. Increasing the concentration to 5 × 10-5 mol/l in an additional series of experiments did not further increase the electrical response to the adrenergic agonist (n = 6, data not shown). Therefore, a concentration of 5 × 10-6 mol/l was chosen for all subsequent experiments.


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Fig. 2.   Concentration-response curve for the first (maximal increase in Isc) and second (maximal decrease in Isc) phase of the effect of epinephrine (administered at the serosal side) in the distal () and proximal colon (). Epinephrine was administered in increasing concentrations to the same tissue with an intermediate washing step between the individual administrations; therefore, a possible desensitization cannot be ruled out. Values are means (symbols) ± SE (error bars); n = 6-8. The threshold at which epinephrine induced a first significant change in Isc amounted to 5 × 10-8 mol/l in the distal colon (for both phases) and 5 × 10-7 mol/l in the proximal colon (for both phases).

When epinephrine was administered three times to the same tissue with an intermediate washing step between the individual administrations, the Isc response decreased from administration to administration, although this desensitization reached statistical significance only in the case of the distal colon (n = 6, data not shown). Therefore, for all further experiments, epinephrine was administered only once to the same preparation.

Ionic nature of the Isc response. Ion substitution experiments were performed to investigate the ionic basis of the biphasic Isc response induced by epinephrine. The increase in Isc was totally dependent on the presence of Cl- in both colonic segments (Fig. 3, Table 2). When NaCl was substituted by sodium gluconate on both sides of the tissue, epinephrine (5 × 10-6 mol/l at the serosal side) did not increase Isc, whereas the decrease in Isc induced by the adrenergic agonist was still present. In the absence of Cl-, the decrease in Isc developed even faster than in the presence of this anion; the half time for the decrease in Isc amounted to 52 ± 5 s (P < 0.05 vs. the half time in Cl--containing buffer; n = 8) in the distal and 92 ± 23 s in the proximal colon (P < 0.05 vs. the half time in Cl--containing buffer; n = 8). In contrast, the inhibitor of the Na+-K+-2Cl- cotransporter, bumetanide (10-4 mol/l at the serosal side), inhibited both phases of the epinephrine response (Table 2). These results are compatible with the assumption that the first phase of the epinephrine-induced increase in Isc is due to Cl- secretion. Bumetanide itself led to a decrease in Isc and Gt in both colonic segments (Table 3).


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Fig. 3.   Effects of epinephrine (5 × 10-6 mol/l at the serosal side) on Isc in the distal (A) and proximal (B) rat colon in the absence of Cl-. Tracings are representative of 8 experiments with each tissue; for statistics, see Table 2.

                              
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Table 2.   Effect of anion replacement or transport inhibitors on the response to epinephrine

                              
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Table 3.   Effect of inhibitors on baseline electrical parameters

The involvement of K+ channels in the epinephrine-induced Isc response was investigated by the use of different K+ channel blockers. Quinine (10-3 mol/l at the mucosal side) inhibited the second phase of the Isc response in the proximal colon and even reversed the usual decrease into an increase of Isc in the distal colon (Fig. 4, Table 2), suggesting the induction of K+ secretion via apical quinine-sensitive K+ channels during the late phase of the epinephrine effect. The reason for the paradox, the long-lasting increase in Isc evoked by epinephrine in the presence of quinine, is unknown. In contrast, TEA (5 × 10-3 mol/l at the mucosal side) was ineffective (Table 2).


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Fig. 4.   Effects of epinephrine (5 × 10-6 mol/l at the serosal side) on Isc in the distal (A) and proximal (B) rat colon in the presence of quinine (10-3 mol/l at the mucosal side). Tracings are representative of 5-7 experiments with each tissue; for statistics, see Table 2.

Unidirectional fluxes of Na+ and Cl- were measured to study the effect of epinephrine on the transport of both ions. The flux measurements were performed 5 min after administration of epinephrine, i.e., during the second phase of the Isc response induced by the adrenergic agonist (Fig. 5). Under control conditions, both in the distal and in the proximal colon, the mucosal-to-serosal fluxes (Jmright-arrow s) of Na+ and Cl- exceeded the corresponding serosal-to-mucosal fluxes (Jsright-arrow m), leading to a net absorption of both ions (Table 4). Epinephrine (5 × 10-6 mol/l) induced a significant decrease of Clmright-arrow s and Clsright-arrow m in the distal but not in the proximal colon (Table 4). In parallel, there was a significant increase in Nasright-arrow m in this colonic segment.


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Fig. 5.   Averaged Isc (A and B) and tissue conductance (Gt; C and D) in distal (A and C) and proximal (B and D) colon during measurement of unidirectional Na+ and Cl- fluxes. Values are means (lines) ± SE (shaded area); n = 16.

                              
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Table 4.   Effect of epinephrine on unidirectional and net fluxes of Na+ and Cl- in the distal and proximal colon

Unidirectional fluxes of 86Rb+ were measured as a marker for K+ transport (11). In both colonic segments, Rbnet was not significantly different from zero (Table 5). Epinephrine increased Rbsright-arrow m in both colonic segments (P < 0.05), i.e., it stimulated K+ secretion.

                              
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Table 5.   Effect of epinephrine on unidirectional and net fluxes of Rb+ in the distal and proximal colon

The alkalinization of an unbuffered solution at the mucosal side of the tissue was used to indirectly measure the effect of epinephrine on HCO-3 secretion (24). The experiments were performed in the presence of bumetanide (10-4 mol/l at the serosal side) to eliminate the effects of epinephrine on Cl- secretion. Under control conditions, there was a spontaneous alkalinization of the mucosal compartment, which, under the assumption that this alkalinization completely represents HCO-3 secretion, gave a transport rate of HCO-3 in the range of 2.5-5 µmol · h-1 · cm-2 at the start of the experiment (Fig. 6). Under control conditions, HCO-3 secretion gradually declined during the time of measurement. This time course was not altered by administration of epinephrine (5 × 10-6 mol/l at the serosal side). The validity of the method was verified by comparing the effect of epinephrine with that of forskolin, which induced a strong HCO-3 secretion measured with the identical protocol (20).


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Fig. 6.   Serosal-to-mucosal flux of HCO-3 (J HCO3sright-arrow m, determined titrimetrically) in the absence () and presence ( ) of epinephrine (5 × 10-6 mol/l) in the distal (A) and proximal (B) rat colon. Data are means (symbols) ± SE (bars); n = 11-15.

Involvement of subepithelial structures in the epinephrine-induced effect on Isc. Indomethacin (10-6 mol/l at the mucosal and the serosal side), a cyclooxygenase inhibitor, abolished the first phase, i.e., the Isc increase, of the epinephrine response in the distal colon, whereas the second phase, i.e., the subsequent decrease in Isc, was unaffected (Fig. 7, Table 6). In contrast, indomethacin was ineffective in the proximal colon. Conversely, pretreatment with the neuronal toxin, TTX (10-6 mol/l at the serosal side) had no effect on the first phase but abolished the second phase of the epinephrine response in the distal colon; the blocker, however, was ineffective in the proximal colon (Fig. 7, Table 6). Consequently, the first phase of the epinephrine effect is mediated by prostaglandins and the second phase by enteric neurons in the distal but not in the proximal colon.


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Fig. 7.   Effects of epinephrine (5 × 10-6 mol/l at the serosal side) on Isc in the distal (A and C) and proximal (B and D) rat colon in the presence of indomethacin (10-6 mol/l at the mucosal and the serosal side; A and B) or in the presence of TTX (10-6 mol/l at the serosal side; C and D). Tracings are representative of 6 experiments with each tissue; for statistics, see Table 5.

                              
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Table 6.   Mediation of the response to epinephrine by prostaglandins and enteric neurons

To further characterize the neuronal component of the epinephrine response in the distal colon, the effects of cholinergic antagonists were tested. Pretreatment of the tissue with the nicotinic antagonist hexamethonium (10-5 mol/l at the serosal side) significantly inhibited the epinephrine-induced decrease in Isc in the distal colon (Table 6), whereas the first phase was unaltered. Atropine (10-6 mol/l at the serosal side), the inhibitor of muscarinic receptors, was ineffective (Table 6). As should be expected from the results with TTX, none of the inhibitors affected the response to epinephrine in the proximal colon (Table 6).

Characterization of the adrenergic receptors mediating the epinephrine effect. The nonselective alpha -receptor blocker phentolamine (10-4 mol/l at the serosal side; see Ref. 4 for references concerning the adrenoceptor blockers) did not inhibit the effect of epinephrine in the distal colon. Instead, a paradoxical increase in the first phase of the epinephrine-induced Isc response was observed (Table 7); a similar phenomenon has already been reported for the rabbit ileum (10). In contrast, in the proximal colon, phentolamine inhibited the second phase of the epinephrine effect while leaving the first phase unaltered. The effect of phentolamine was mimicked by the alpha 2-receptor blocker yohimbine (10-5 mol/l at the serosal side; Fig. 8, Table 7) in the proximal colon but not by the alpha 1-antagonist, prazosin (10-6 mol/l at the serosal side; Table 7), suggesting a mediation of the epinephrine response by alpha 2-receptors in the proximal colon. None of these inhibitors was effective in the distal colon (Table 7).

                              
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Table 7.   Effect of epinephrine on Isc in the absence and presence of adrenergic receptor blockers


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Fig. 8.   Effects of epinephrine (5 × 10-6 mol/l at the serosal side) on Isc in the distal (A and C) and proximal (B and D) rat colon in the presence of the alpha 2-receptor blocker yohimbine (10-5 mol/l at the serosal side; A and B) or in the presence of the beta 2-receptor blocker ICI-118551 (10-5 mol/l at the serosal side; C and D). Tracings are representative of 6 experiments with each tissue; for statistics, see Table 6.

The second phase of the epinephrine effect was inhibited in the distal colon by pretreatment of the tissue with the nonselective beta -receptor blocker, propranolol (5 × 10-6 at the serosal side; Table 7). Inhibition was mimicked by the beta 2-receptor blocker, ICI-118551 (10-5 mol/l at the serosal side; Fig. 8); the specific beta 1-receptor blocker atenolol (10-4 mol/l at the serosal side) was ineffective (Table 7), indicating the involvement of beta 2-receptors in the mediation of the epinephrine effect in the distal colon. None of the beta -receptor blockers was effective in the proximal colon (Table 7).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Epinephrine induces a biphasic change of Isc in the rat colon, which consists of an initial, transient increase followed by a long-lasting decay (Fig. 1). The first phase of the action of epinephrine is caused by a Cl- secretion, as indicated by anion substitution experiments and by the sensitivity to bumetanide, an inhibitor of the basolateral Na+-K+-2Cl- cotransporter (Fig. 3, Table 2). Depending on the individual colonic segment, only 70-79% of the tissues investigated exhibited a rapid increase in Isc. Such a variability was already observed by Racusen and Binder (17) for the rat colon. The ability of epinephrine to induce Cl- secretion seems to be species dependent: it is absent in rabbit ileum (10) and in rabbit colon (22) but present in guinea pig colon (18).

In contrast, the second phase of the epinephrine response could be regularly evoked in more than 90% of the tissues. The decrease in Isc was suppressed by apical administration of the K+ channel blocker quinine (Fig. 4) and was paralleled by an increase in Rbsright-arrow m (Table 5), indicating that epinephrine induces a K+ secretion, a response that has already been observed in several other intestinal epithelia such as rabbit (12, 22) or guinea pig colon (18). The decrease in Isc was suppressed by bumetanide but not in the absence of Cl- (Table 2). At first glance, this paradoxical result may be explained by the fact that the Cl--poor buffer still contained Cl- due to the presence of K+ and Ca2+ salts of this anion; obviously, this Cl- is sufficient to allow K+ influx via the Na+-K+-2Cl- cotransporter.

The K+ secretion begins earlier than the observed decrease in Isc. Under Cl--poor conditions, i.e., under conditions in which epinephrine is not able to induce Cl- secretion, the half time for the development of the decrease in Isc is significantly shortened, suggesting that under control conditions the effect of the induction of K+ secretion, which leads to a decrease in Isc, is partially counteracted by the induction of Cl- secretion leading to an increase in Isc. Except for a decrease in Clsright-arrow m in the distal colon, representing an inhibition of spontaneous Cl- secretion, which was accompanied by a (smaller) decrease in Clmright-arrow s (Table 4), no relevant changes in the transport of Na+ and Cl- (Table 4) or the transport of HCO-3 (Fig. 6) were observed during the long-lasting decrease in Isc induced by epinephrine. This raises the question concerning the nature of the part of the Isc response, which is several times greater than the change in net K+ transport (Table 5), independent of K+ secretion. This "unexplained Isc" might represent either changes in the electrogenic transport of other ions present in the buffer solution or, more probably, the combinations of subtle changes in Na+, Cl-, and HCO-3 transport not resolved in the corresponding flux studies.

Despite the phenomenologically similar action of epinephrine on Isc in the distal and the proximal colon (cf. Fig. 1, A and B, respectively), there are characteristic differences in the mediation of the response to the adrenergic agonist. In the distal colon, both phases of the effect of epinephrine are mediated by extraepithelial action sites. In this segment, the first phase of the epinephrine-induced change in Isc is suppressed by the cyclooxygenase inhibitor, indomethacin (Fig. 7), indicating the involvement of prostaglandins in the induction of Cl- secretion. The predominant part of prostaglandin synthesis in the rat colon takes place in the connective tissue of the submucosa (6), suggesting an effect of the adrenergic agonist on subepithelial structures. In contrast, the second phase of the epinephrine response was insensitive to indomethacin but was abolished by the neurotoxin TTX (Fig. 7) in the distal colon, indicating that the drug stimulates secretomotoneurons of the enteric nervous system, which induce K+ secretion at the epithelium.

The nature of the transmitter finally mediating the secretory signal to the colonocyte is not known; an involvement of ACh can be excluded by the missing effect of atropine on the epinephrine-induced decrease in Isc (Table 6). Obviously, a nicotinic synapse is involved in the neuronal secretory pathway, as shown by the partial sensitivity to the nicotinic antagonist, hexamethonium (Table 6). In contrast, none of these inhibitors was effective in the proximal colon, suggesting a direct epithelial site of action of epinephrine. At first glance, these results seem to be in conflict with previous observations in rat colon, in which the decrease in Isc evoked by epinephrine was resistant to a somewhat lower concentration (2 × 10-7 mol/l) of TTX (17). However, in this study, no distinction was made between the distal and the proximal colon, which may easily explain the apparent discrepancy. In other species such as rabbit (22) or guinea pig colon (18), the decrease in Isc evoked by epinephrine is TTX insensitive.

Different receptors are involved in the epithelial and nonepithelial actions of epinephrine in both colonic segments. In the proximal colon, the decrease in Isc evoked by epinephrine did not happen in the presence of phentolamine, a nonselective alpha -blocker (Table 7), and in the presence of yohimbine, a selective alpha 2-blocker (Fig. 8), suggesting the involvement of alpha 2-receptors in the induction of K+ secretion. A similar sensitivity to adrenoceptor blockers has been observed in rabbit ileum (5) in which yohimbine abolishes the decrease in Isc evoked by epinephrine. In contrast, in the rabbit colon, the TTX-resistant K+ secretion is mediated by a beta 1-receptor (22).

In the distal colon, the TTX-sensitive decrease in Isc evoked by epinephrine was suppressed by the nonselective beta -blocker, propranolol (Table 7), and by the selective beta 2-blocking drug, ICI-118551 (Fig. 8), indicating mediation by a beta 2-receptor. Neuronal effects of epinephrine on enteric neurons have been described, although they are in general attributed to the stimulation of alpha 2-receptors (16). However, beta -receptors on other nerve structures are well known (see e.g., Ref. 23); therefore, the most simple explanation of these data is the assumption of a beta -receptor on an enteric neuron, although a localization on another cell type, which then triggers secretomotoneurons, is not excluded.

Surprisingly, the first phase of the epinephrine response was not inhibited in either the distal or the proximal colon by any of the tested alpha - or beta -blockers (Table 7). The reason for this failure is unknown. There is, however, the speculative possibility that this phase of the action of epinephrine may be mediated by another type of adrenoceptor, e.g., beta 3-receptors as observed in fatty tissue or intestinal smooth muscle (for references, see Ref. 3), which are in general quite resistant against even nonselective beta -blockers such as propranolol. Because of the transient (and variable) nature of this part of the epinephrine response, no further studies were performed to clarify this point.

We can only speculate about the nature of the second messenger(s) involved in the induction of K+ secretion in both colonic segments. An increase in both the intracellular Ca2+ as well as in the intracellular cAMP concentration induces K+ secretion by different mechanisms (8, 19). alpha 2-Receptors, which mediate the action of epinephrine on K+ transport in the proximal colon and which are most likely located at the epithelium, are known to be coupled to an increase in the intracellular Ca2+ concentration as well as an inhibition of adenylate cyclase (for references, see Ref. 13); therefore, it may be possible that Ca2+ is the second messenger inducing the K+ secretion in the proximal colon. The neurotransmitter, which is released by the secretomotoneurons stimulated by epinephrine in the distal colon, is not known; therefore, all hypotheses about the intracellular mediation of the epinephrine response in this colonic segment remain speculative.

Taken together, these data indicate a segment-specific regulation of transepithelial ion transport in rat colon by adrenergic stimuli: a direct epithelial action of epinephrine in the proximal colon and an indirect action in the distal colon, mediated by the enteric nervous system in the case of epinephrine-induced K+ secretion and mediated by prostaglandins in the case of epinephrine-induced Cl- secretion. Thus colonic K+ transport is, in addition to Cl- transport (7), a further example of several elements of the intestinal wall, i.e., the eicosanoid-producing submucosal tissue, the enteric nervous system, and the ion-transporting cell (i.e., the epithelium), showing a strong interaction in the regulation of intestinal functions.

    ACKNOWLEDGEMENTS

We are grateful for the diligence of B. Brück, E. Haas, A. Metternich, and B. Schmitt.

    FOOTNOTES

This work was supported by Deutsche Forschungsgemeinschaft Grant Di 388/3-1.

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: M. Diener, Institut für Veterinär-Physiologie, Universität Giessen, Frankfurter Str. 100, D-35392 Giessen, Germany.

Received 21 April 1998; accepted in final form 5 August 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(6):G1367-G1376
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