Stimulation of Na⁺‐K⁺‐pump currents by epithelial nicotinic receptors in rat colon

Abstract

Background and Purpose

Acetylcholine‐induced epithelial Cl⁻ secretion is generally thought to be mediated by epithelial muscarinic receptors and nicotinic receptors on secretomotor neurons. However, recent data have shown expression of nicotinic receptors by intestinal epithelium and the stimulation of Cl⁻ secretion by nicotine, in the presence of the neurotoxin, tetrodotoxin. Here, we aimed to identify the transporters activated by epithelial nicotinic receptors and to clarify their role in cholinergic regulation of intestinal ion transport.

Experimental Approach

Ussing chamber experiments were performed, using rat distal colon with intact epithelia. Epithelia were basolaterally depolarized to measure currents across the apical membrane. Apically permeabilized tissue was also used to measure currents across the basolateral membrane in the presence of tetrodotoxin.

Key Results

Nicotine had no effect on currents through Cl⁻ channels in the apical membrane or on currents through K⁺ channels in the apical or the basolateral membrane. Instead, nicotine stimulated the Na⁺‐K⁺‐pump as indicated by Na⁺‐dependency and sensitivity of the nicotine‐induced current across the basolateral membrane to cardiac steroids. Effects of nicotine were inhibited by nicotinic receptor antagonists such as hexamethonium and mimicked by dimethyl‐4‐phenylpiperazinium, a chemically different nicotinic agonist. Simultaneous stimulation of epithelial muscarinic and nicotinic receptors led to a strong potentiation of transepithelial Cl⁻ secretion.

Conclusions and Implications

These results suggest a novel concept for the cholinergic regulation of transepithelial ion transport by costimulation of muscarinic and nicotinic epithelial receptors and a unique role of nicotinic receptors controlling the activity of the Na⁺‐K⁺‐ATPase.

Abbreviations

DhβE

dihydro‐β‐erythroidine

DMPP

dimethyl‐4‐phenylpiperazinium

I_sc

short‐circuit current

TTX

tetrodotoxin

Tables of Links

TARGETS
Ligand‐gated ion channels a
Nicotinic acetylcholine receptors
GPCRs b
Muscarinic acetylcholine receptors
Transporters c
Na+/K+ ATPases

LIGANDS
Atropine	Mecamylamine
α‐Bungarotoxin	Ouabain
Dihydro‐β‐erythroidine	Strychnine
Forskolin	Tetrodotoxin
Hexamethonium

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (^a,b,cAlexander et al., 2015a,b,c).

Introduction

Acetylcholine is a classical neurotransmitter in the peripheral and central nervous systems. In the intestine, the main source of neuronal acetylcholine is the enteric nervous system formed by the submucosal and myenteric plexus. Two classes of receptors mediate the action of acetylcholine: G protein‐coupled muscarinic receptors, of which five subtypes (M₁ to M₅) are known (Caulfield and Birdsall, 1998), and different types of homo‐ or heteropentameric nicotinic receptors, which, in general, function as ligand‐gated ion channels (Albuquerque et al., 2009). It is generally thought that nicotinic receptors mediate the effect of acetylcholine on excitable tissues such as neurons or skeletal muscle. In contrast, muscarinic receptors are observed pre‐ and postsynaptically in neurons, and they mediate the action of this transmitter in organs or tissues innervated by the autonomous nervous system. For example, in the gut, M₁ receptors are found on epithelial cells (Haberberger et al., 2006, Wessler and Kirkpatrick, 2008) and on enteric neurons (North et al., 1985), M₂ receptors on smooth muscle cells (Bhattacharya et al. 2013) and M₃ receptors on epithelial and smooth muscle cells (Wessler and Kirkpatrick, 2008).

This classical view of cholinergic signal transduction has been challenged by recent observations. For example, non‐neuronal cells, mainly with barrier or defence function, that is, epithelial and immune cells, also synthesize and release acetylcholine (Wessler et al., 2003, Wessler and Kirkpatrick, 2008). Also, rat colonic surface epithelium synthesizes acetylcholine and releases it as a paracrine mediator after contact with the short‐chain fatty acid propionate (Yajima et al., 2011) via transporters of the organic cation transporter family (Bader et al., 2014). Besides acetylcholine, this tissue also synthesizes other choline esters such as propionylcholine or butyrylcholine, which stimulate cholinergic receptors and contribute to local signalling within colonic epithelium (Moreno et al., 2016).

A further modification of the textbook view on cholinergic signalling results from the expression of nicotinic receptors in non‐excitable tissue such as epithelia. For example, in murine tracheal epithelium, nicotinic receptors are involved in the regulation of the production of airway lining fluid (Hollenhorst et al., 2012). Also, colonic epithelium expresses – besides muscarinic receptors of the M₁ and the M₃ subtype (Haberberger et al., 2006, Bader and Diener, 2015) – different subunits of nicotinic receptors (α2, α4, α5, α6, α7, α10 and β4), and stimulation of these receptors with nicotinic agonists induces a Cl⁻ secretion (Bader and Diener, 2015). In contrast to stimulation of muscarinic receptors, which activates anion secretion via hyperpolarization of the membrane due to opening of basolateral (and apical) Ca²⁺‐dependent K⁺ channels (Strabel and Diener, 1995) and transient stimulation of apical Ca²⁺‐dependent Cl⁻ channels (Hennig et al., 2008), nothing is known about the mechanism of action of nicotinic receptors in intestinal epithelium. Therefore, in the present study, we measured transepithelial currents and selective currents across the apical or the basolateral membrane in rat distal colon in order to identify the ion transporters, which are activated by nicotinic receptors and their potential interaction with muscarinic receptors during regulation of transepithelial transport.

Methods

Animals

All animal care and experimental procedures were approved by the named animal welfare officer of the Justus Liebig University (administrative number 577_M) and performed according to the German and European animal welfare law. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015).

Wistar rats (160–250 g) were used in these experiments. Both female and male rats were used in about equal number (overall 45% males, 55% females) with random distribution of the sex of the used animals within the individual experimental groups. The animals were bred and housed at the Institute of Veterinary Physiology and Biochemistry of the Justus‐Liebig‐University with free access to food and water until the time of experiment. Animals were housed in macrolone type IV cages with three animals per cage at 22°C, 50% air humidity and a light : dark cycle of 12:12 h. Animals were killed after CO₂ narcosis, by cervical dislocation followed by exsanguination.

Solutions

If not indicated otherwise (as experiments with apically permeabilized or basolaterally depolarized epithelia), Ussing chamber experiments were carried out in a bathing solution of the following composition (mmol·L⁻¹): 107 NaCl, 4.5 KCl, 25 NaHCO₃, 1.8 Na₂HPO₄, 0.2 NaH₂PO₄, 1.25 CaCl₂, 1 MgSO₄, 12.2 glucose. The solution was gassed with 5% (v/v) CO₂ and 95% (v/v) O₂ at 37°C and had a pH of 7.4 (adjusted by NaHCO₃/HCl). For the Cl⁻‐free buffer, NaCl and KCl were equimolarly substituted by Na gluconate (NaGluc) and K gluconate (KGluc); CaCl₂ was replaced by Ca gluconate (CaGluc) in a concentration of 5.75 mmol·L⁻¹ (in order to compensate the Ca²⁺‐buffering properties of gluconate).

For the depolarization of the basolateral membrane, a 111.5 mmol·L⁻¹ KCl solution was used in the serosal compartment, in which NaCl was equimolarly replaced by KCl. In order to drive a Cl⁻ current across apical Cl⁻ channels under these conditions, the mucosal solution contained 111.5 mmol·L⁻¹ K gluconate (KGluc) instead of NaCl and KCl.

In several experiments with apically permeabilized epithelia, a K⁺ gradient was applied from the mucosal to the serosal side by increasing the KCl concentration in the standard HCO₃ ⁻‐buffered solution to 13.5 mmol·L⁻¹ in the mucosal compartment, while reducing the NaCl concentration to 98 mmol·L⁻¹ in order to maintain iso‐osmolarity. For the Na⁺‐free solution, NaCl was replaced by N‐methyl‐D‐glucamine (NMDG⁺) Cl‐.

Tissue preparation

Rat distal colon, which is a well‐established model to study epithelial anion secretion, was quickly removed and placed in ice‐cold Ussing chamber bathing solution. The large intestine was carefully flushed several times before it was mounted on a thin plastic rod. A circular incision was made near the distal end with a blunt scalpel. The serosa and muscularis propria were stripped off by hand in order to obtain a mucosa‐submucosa preparation. Two segments from distal colon were prepared from each animal; in general, one segment served as control to study the response to a nicotinic agonist in the absence and the other segment to study the response in the presence of putative inhibitors. Segments were randomly distributed by a technician; blinding did not seem to be appropriate for these in vitro experiments.

Ussing chamber experiments

The mucosa‐submucosa preparations were fixed in a modified Ussing chamber, bathed with a volume of 3.5 mL on each side. The tissue was incubated at 37°C and short‐circuited by a computer‐controlled voltage‐clamp device (Ingenieur Büro für Mess‐ und Datentechnik Mussler, Aachen, Germany) with correction for solution resistance. The exposed surface of the tissue was 1 cm². The electrodes used for voltage measurement and current application were Ag/AgCl electrodes in 3 mol·L⁻¹ KCl, which were separated from the chamber lumen by agar bridges (46.7 g·L⁻¹ agar in the standard bathing solution). Short‐circuit current (I_sc) was continuously recorded, and tissue conductance (G_t) was measured every minute by applying a current pulse of ±50 μA·cm⁻² with a duration of 200 ms. I_sc is expressed as μEq·h⁻¹·cm⁻², that is, the flux of a monovalent ion per time and area with 1 μEq·h⁻¹·cm⁻² = 26.9 μA·cm⁻². Substances were administered after an equilibration period of about 60 min. All experiments were performed in the presence of tetrodotoxin (TTX; 10⁻⁶ mol·L⁻¹ at the serosal side) in order to prevent actions of nicotinic receptor stimulation on enteric neurons by blocking voltage‐dependent Na⁺ channels with this neurotoxin (Catterall, 1980).

The maximal increase in I_sc evoked by an agonist is given as the difference from the baseline value, just prior administration of the drug. In those experiments, where the I_sc did not stabilize, that is, the administration of drugs during the decaying phase of the nystatin‐induced I_sc, the theoretical course of I_sc was calculated by linear regression analysis as described previously (Schultheiss and Diener, 1997). To do so, the I_sc 3 min prior administration of the drug (30 data points, as I_sc was registered every 6 s) was used to calculate the regression line. This regression served to extrapolate the decay of I_sc in the absence of nicotine, which was subtracted from the maximal increase in I_sc evoked by nicotine.

When a drug (which evoked reproducible effects after repeated administration) was administered repeatedly to the same tissue (as in the experiments shown in Figure 6B), the compartment to which the substance had been administered was washed three times with 5× the chamber volume, before the drug was administered again.

Figure 6.

(A) Increase transepithelial chloride secretion measured as increase in I_sc (ΔI_sc) in intact epithelia evoked by nicotine (10⁻⁶ mol·L⁻¹; green bar), pilocarpine (4·10⁻⁶ mol·L⁻¹; blue bar) and the combination of both agonists (red bar). Statistically homogenous groups are marked by the same letter (ANOVA followed by post hoc Tukey's test). (B) Effect of nicotine (10⁻⁶ mol·L⁻¹; green symbols), different concentrations of pilocarpine (from 2 to 50·10⁻⁶ mol·L⁻¹; blue symbols) and the combination of both (red symbols) on I_sc. For graphical clarity, two of the three data symbols for the same concentration(s) were laterally shifted to the right or the left, respectively, in order to avoid an overlay of the data points. The agonists were administered to the serosal side. All experiments were performed in the presence of TTX (10⁻⁶ mol·L⁻¹ at the serosal side). Values are given as differences from baseline I_sc (ΔI_sc) just before administration of drugs and are means ± SEM; for n, see table below Figure 6B.

Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). Results are given as mean ± SEM with the number (n) of investigated tissues. For all Ussing chamber experiments, a group size of n = 6–7 was designed; in some experimental series, it was increased up to nine, if there was a larger variability. For the comparison of two groups, either Student's t‐test or Mann–Whitney‐U‐test was applied. An F‐test decided which test method had to be used. Both paired and unpaired two‐tailed Student's t‐tests were applied as appropriate. When means of more than two groups had to be compared, ANOVA was used. If there was no significant variance inhomogeneity and an F‐test indicated that variances between the groups were significantly larger than within the groups, a Tukey post hoc test was performed. P < 0.05 was considered to be statistically significant. Statistical comparisons were performed using the statistical software Winstat 2012.1 (R. Fitch Software, Bad Krozingen, Germany). Linear regressions were calculated with Excel 2010 (Microsoft, Redmond, WA, USA).

Materials

Bumetanide, forskolin and mecamylamine were dissolved in ethanol (final maximal ethanol concentration 0.5 mL·L⁻¹). Scilliroside (gift from Sandoz, Basel, Switzerland) was dissolved in methanol (final methanol concentration 2 mL·L⁻¹). Dihydro‐β‐erythroidine (DhβE; Tocris, Bristol, UK) and ouabain were dissolved in DMSO (final maximal DMSO concentration 1 mL·L⁻¹). Nystatin was dissolved in DMSO (final DMSO concentration 2 mL·L⁻¹); the stock solution was ultrasonicated immediately before use. TTX was dissolved in 2.10⁻² mol·L⁻¹ citrate buffer. Atropine, α‐bungarotoxin (Tocris, Bristol, UK), dimethyl‐4‐phenylpiperazinium (DMPP), hexamethonium, nicotine, pilocarpine and strychnine were dissolved in distilled water. Conotoxin MVIIC (5·10⁻⁸ mol·L⁻¹) and conotoxin SVIB (both from Alomone Labs, Jerusalem, Israel) were dissolved in distilled water containing 1 mg·L⁻¹ bovine serum albumin. If not indicated otherwise, drugs were purchased from Sigma‐Aldrich, Taufkirchen, Germany.

Results

Nicotinic receptors do not activate apical ion channels

As nicotine induces Cl⁻ secretion (Bader and Diener, 2015), which centrally involves Cl⁻ efflux via anion channels in the apical membrane (Barrett and Keely, 2000), we needed to know if stimulation of nicotinic receptors leads to activation of a Cl⁻ conductance in the apical membrane. A chemical permeabilization of the basolateral membrane by ionophores, for example, by nystatin, is not possible in our hands as ionophores do obviously not pass the tissue layers underlying the epithelium in sufficient amounts. Therefore, the basolateral membrane was ‘electrically eliminated’ by depolarization with a buffer containing a high K⁺ concentration. Due to the high basolateral K⁺ permeability, the electrical properties of the tissue, which are normally characterized by two batteries in series, are then dominated by the apical membrane (Fuchs et al., 1977, Schultheiss and Diener, 1997). A Cl⁻ concentration gradient (111.5 mmol·L⁻¹ KCl at the serosal side, 111.5 mmol·L⁻¹ K gluconate at the mucosal side) served as driving force for Cl⁻ flux across apical anion channels (see schematic inset in Figure 1). This series of experiments and all subsequently described experiments were performed in the continuous presence of the neurotoxin TTX (10⁻⁶ mol·L⁻¹ at the serosal side) in order to prevent effects of nicotine mediated by cholinergic receptors on secretomotor neurons of the enteric nervous system. However, under these conditions, nicotine (10⁻⁴ mol·L⁻¹ at the serosal side; for a complete concentration–response curve describing the effect of nicotine on Cl⁻ secretion across rat colonic epithelium, see Bader and Diener, 2015) was completely ineffective (Figure 1). As positive control, at the end of the experiment, the activator of adenylate cyclase(s) forskolin (5·10⁻⁶ mol·L⁻¹ at the mucosal and the serosal side) was administered, which induced a prompt positive I_sc, as expected, when the cystic fibrosis transmemembrane regulator, that is, the dominant anion channel in the apical membrane of colonic epithelium (Verkman and Galietta, 2013), is activated by cAMP‐dependent phosphorylation.

Figure 1.

Missing effect of nicotine (10⁻⁴ mol^.L⁻¹ at the serosal side) in the presence of TTX (10⁻⁶ mol^.L⁻¹ at the serosal side) on current across apical Cl⁻ channels in basolaterally depolarized epithelia (mucosal solution 111.5 mmol·L⁻¹ KGluc; serosal solution: 111.5 mmol·L⁻¹ KCl as indicated by the schematic inset). At the end of the experiment, forskolin (5·10⁻⁶ mol^.L⁻¹ at the mucosal and the serosal side) was administered as positive control. Mucosa‐submucosa preparations from rat distal colon. Values are means ± SEM, n = 8.

Nicotine did also not activate apical K⁺ channels. When the basolateral depolarization with the 111.5 mmol·L⁻¹ KCl buffer at the serosal side was performed in the absence of a Cl⁻ concentration gradient, that is, with the standard 107 mmol·L⁻¹ NaCl/4.5 mmol·L⁻¹ KCl buffer at the mucosal side, nicotine (10⁻⁴ mol·L⁻¹ at the serosal side) did not induce any change in I_sc (n = 6, data not shown). Thus, nicotinic receptors do not act via ion channels in the apical membrane.

Activation of electrogenic ion transport across the basolateral membrane by nicotine

In order to find out whether the site of action of nicotinic receptors is electrogenic ion transporters in the basolateral membrane, the apical membrane was permeabilized by the ionophore nystatin (100 μg·mL⁻¹ at the mucosal side). In a first step, buffer solutions were selected, which allowed both a flux of K⁺ ions across basolateral K⁺ channels by applying a mucosal to serosal K⁺ concentration gradient as well as an activation of the pump current carried by the 3 Na⁺‐2 K⁺‐ATPase that is stimulated when Na⁺ enters the epithelial cells via the nystatin pores (mucosal: 98 mmol·L⁻¹ NaCl/13.5 mmol·L⁻¹ KCl; serosal: 107 mmol·L⁻¹ NaCl/4.5 mmol·L⁻¹ KCl as indicated by the schematic inset in Figure 2). As described previously, nystatin induced a strong stimulation of I_sc under these conditions, which slowly fades over time (Schultheiss and Diener, 1997). When nicotine (10⁻⁴ mol·L⁻¹ at the serosal side) was administered during this decaying phase, it induced a prompt, transient increase in I_sc indicating a stimulation of electrogenic ion transport across the basolateral membrane (Figure 2). In average, nicotine‐stimulated I_sc amounted to 2.0 ± 0.39 μEq·h⁻¹·cm⁻² (n = 8, P < 0.05 vs. extrapolated baseline, see Methods) under these conditions.

Figure 2.

Effect of nicotine (10⁻⁴ mol^.L⁻¹ at the serosal side) in the presence of TTX (10⁻⁶ mol^.L⁻¹ at the serosal side) on total current across the basolateral membrane (mucosal: 98 mmol·L⁻¹ NaCl/13.5 mmol·L⁻¹ KCl; serosal: 107 mmol·L⁻¹ NaCl/4.5 mmol·L⁻¹ KCl as indicated by the schematic inset). The apical membrane was permeabilized by nystatin (100 μg·mL⁻¹ at the mucosal side). Mucosa‐submucosa preparations from rat distal colon. Values are means ± SEM, n = 8.

The nicotine‐evoked current was not caused by an activation of basolateral K⁺ channels. When the apical membrane was permeabilized in the absence of mucosal Na⁺ but in the presence of a mucosal to serosal K⁺ concentration gradient (mucosal 98 mmol·L⁻¹ NMDGCl/13.5 mmol·L⁻¹ KCl; serosal 107 mmol·L⁻¹ NMDGCl/4.5 mmol·L⁻¹ KCl as indicated by the schematic inset in Figure 3), nicotine (10⁻⁴ mol·L⁻¹ at the serosal side) was completely ineffective (Figure 3).

Figure 3.

Missing effect of nicotine (10⁻⁴ mol^.L⁻¹ at the serosal side) in the presence of TTX (10⁻⁶ mol^.L⁻¹ at the serosal side) on current across basolateral K⁺ channels (mucosal 98 mmol·L⁻¹ NMDGCl/13.5 mmol·L⁻¹ KCl; serosal 107 mmol·L⁻¹ NMDGCl/4.5 mmol·L⁻¹ KCl as indicated by the schematic inset). The apical membrane was permeabilized by nystatin (100 μg·mL⁻¹ at the mucosal side). Mucosa‐submucosa preparations from rat distal colon. Values are means ± SEM, n = 8.

In contrast, in the presence of mucosal Na⁺, but the absence of a K⁺ concentration gradient, that is, in the absence of a driving force for K⁺ flux across K⁺ channels (107 mmol·L⁻¹ NaCl/4.5 mmol·L⁻¹ KCl on both sides of the chamber, see schematic inset in Figure 4), nicotine (10⁻⁴ mol·L⁻¹ at the serosal side) induced a strong increase in I_sc across the basolateral membrane in apically permeabilized epithelia (Figure 4A). The nicotine‐induced current was nearly suppressed in the presence of scilliroside (10⁻⁴ mol·L⁻¹ at the serosal side; Figure 4B, Table 1), which effectively blocks the Na⁺‐K⁺‐ATPase from rats (Robinson, 1970). A weaker, but statistically significant, inhibition was also observed, when the epithelium was pretreated with ouabain (Table 1), the prototypical inhibitor of the Na⁺‐K⁺‐pump, for which the rat α₁ subunit [that forms – together with a β₁ subunit – the dominant form of this transport enzyme in rat intestine (Escoubet et al., 1997)] is known to be less sensitive in comparison with other species (Blanco and Mercer, 1998). Taken together, the cation substitution experiments combined with these blocker experiments clearly indicate that the nicotine‐induced I_sc is carried by the basolateral Na⁺‐K⁺‐ATPase.

Figure 4.

Effect of nicotine (10⁻⁴ mol^.L⁻¹ at the serosal side) in the presence of TTX (10⁻⁶ mol^.L⁻¹ at the serosal side) on current mediated by the Na⁺‐K⁺‐pump (mucosal and serosal solution: 107 mmol·L⁻¹ NaCl/4.5 mmol·L⁻¹ KCl as indicated by the schematic inset). The effect of nicotine was tested on the presence of methanol (the solvent for scilloride, A) or in the presence of scilliroside (10⁻⁴ mol·L⁻¹ at the serosal side, B). The apical membrane was permeabilized by nystatin (100 μg·mL⁻¹ at the mucosal side). Mucosa‐submucosa preparations from rat distal colon. Values are means ± SEM, n = 8 (each group), *P < 0.05, significantly different from extrapolated baseline.

Table 1.

Sensitivity of nicotine‐induced current to cardiac steroids

	Nicotine‐induced pump current Δ Isc (μEq·h−1·cm−2)		n
Inhibitor	Without inhibitor	With inhibitor
Scilliroside	3.58 ± 0.56	0.66 ± 0.19*	8, 8
Ouabain	5.26 ± 0.62	2.30 ± 0.40*	8, 8

Effect of nicotine (10⁻⁴ mol·L⁻¹ at the serosal side) on the current across the basolateral membrane carried by the Na⁺‐K⁺‐pump, with or without inhibitors of the Na⁺‐K⁺‐ATPase. The apical membrane was permeabilized with nystatin (100 μg·mL⁻¹ at the mucosal side). The mucosal and the serosal solution was 107 mmol·L⁻¹ NaCl/4.5 mmol·L⁻¹ KCl (i.e. a missing K⁺ concentration gradient prevented currents across basolateral K⁺ channels). Concentrations of inhibitors (all administered to the serosal side) were as follows: scilliroside (10⁻⁴ mol·L⁻¹) and ouabain (10⁻⁴ mol·L⁻¹). Values are given as differences from the extrapolated baseline current after administration of nystatin (ΔI_sc; see Methods) and are means ± SEM.

^* P < 0.05, significantly different from ΔI_sc without the inhibitor

Characterization of the receptors involved in the stimulation of pump current induced by nicotine

In order to investigate, whether the action of nicotine on pump current is indeed mediated by nicotinic receptors, the colonic epithelium was pretreated with hexamethonium (10⁻⁴ mol·L⁻¹ at the serosal side), a non‐selective inhibitor of nicotinic receptors. Under these conditions, the nicotine‐induced I_sc was nearly suppressed (Table 2). A weaker, but statistically significant, inhibition was observed in the presence of another inhibitor of nicotinic receptors, mecamylamine (10⁻⁴ mol·L⁻¹ at the serosal side; Table 2). Vice versa, DMPP (10⁻⁴ mol·L⁻¹ at the serosal side), a chemically different nicotinic agonist, stimulated the current carried by the Na⁺‐K⁺‐pump (Figure 5A), an effect, which was suppressed by hexamethonium (10⁻⁴ mol·L⁻¹ at the serosal side; Figure 5B).

Table 2.

Sensitivity of nicotine‐induced current to different inhibitors

	Nicotine‐induced pump current Δ Isc (μEq·h−1·cm−2)		n
Inhibitor	Without inhibitor	With inhibitor
Hexamethonium	6.43 ± 1.39	0.35 ± 0.12*	6, 6
Mecamylamine	3.65 ± 0.46	2.15 ± 0.40*	8, 7
Strychnine	3.59 ± 0.65	2.26 ± 0.59	6, 6
α‐Bungarotoxin	3.02 ± 1.00	3.96 ± 0.80	8, 7
DhβE	4.78 ± 0.30	3.07 ± 0.50*	8, 9
Atropine	4.50 ± 0.59	2.42 ± 0.23*	7, 8
DhβE + atropine	3.72 ± 0.58	1.50 ± 0.46*	7, 8
DhβE + atropine + strychnine	4.51 ± 1.05	2.16 ± 0.43*	6, 8
Conotoxin MVIIC + SVIB	4.46 ± 1.03	6.26 ± 1.03	5, 6
DMSO	3.24 ± 0.84	3.98 ± 0.84	6, 6
Ethanol	6.32 ± 1.23	4.93 ± 1.06	6, 6

Effect of nicotine (10⁻⁴ mol^.L⁻¹ at the serosal side) on the current across the basolateral membrane carried by the Na⁺‐K⁺‐ATPase, with or without antagonists of nicotinic receptors. The apical membrane was permeabilized with nystatin (100 μg·mL⁻¹ at the mucosal side). The mucosal and the serosal solution was 107 mmol·L⁻¹ NaCl/4.5 mmol·L⁻¹ KCl (i.e. a missing K⁺ concentration gradient prevented currents across basolateral K⁺ channels). Concentrations of inhibitors (all administered to the serosal side) were as follows: hexamethonium (10⁻⁴ mol·L⁻¹), mecamylamine (10⁻⁵ mol·L⁻¹), strychnine (10⁻⁵ mol·L⁻¹), α‐bungarotoxin (10⁻⁶ mol·L⁻¹), DhβE (10⁻⁵ mol·L⁻¹), atropine (2.5 x 10⁻⁵ mol·L⁻¹), conotoxin MVIIC (5 x 10⁻⁸ mol·L⁻¹) and conotoxin SVIB (5 x·10⁻⁸ mol·L⁻¹). Furthermore, control experiments with the highest concentrations of the solvents DMSO (1 mL·L⁻¹) and ethanol (0.5 mL·L⁻¹) are shown. Values are given as difference to the extrapolated baseline current after administration of nystatin (ΔI_sc; see Methods) and are means ± SEM.

^* P < 0.05 significantly different from ΔI_sc without inhibitor.

Figure 5.

Effect of DMPP (10⁻⁴ mol^.L⁻¹ at the serosal side) in the presence of TTX (10⁻⁶ mol^.L⁻¹ at the serosal side) on current mediated by the Na⁺‐K⁺‐pump (mucosal and serosal: 107 mmol·L⁻¹ NaCl/4.5 mmol·L⁻¹ KCl as indicated by the schematic inset). The effect of DMPP was tested on the absence (A) and presence (B) of hexamethonium (10⁻⁴ mol·L⁻¹ at the serosal side). The apical membrane was permeabilized by nystatin (100 μg·mL⁻¹ at the mucosal side). Mucosa‐submucosa preparations from rat distal colon. Values are means ± SEM; n = 7 (each group), *P < 0.05, significantly different from extrapolated baseline.

As several subunits of nicotinic receptors have been detected on the mRNA level in rat colonic epithelium (Bader and Diener, 2015), which can be thought to form different combinations of functional nicotinic receptors, experiments with ‘bona fide’ subtype selective blockers of nicotinic receptors were performed (for references to the inhibitors used, see Chavez‐Noriega et al., 1997, Wonnacott and Barik, 2007). In the presence of strychnine (10⁻⁵ mol·L⁻¹ at the serosal side), which besides its well‐known action on glycine receptors also blocks α7, α8 and α9α10‐subunits of nicotinic receptors, the pump current induced by nicotine (10⁻⁴ mol·L⁻¹ at the serosal side) tended to be reduced, although this effect did not reach statistical significance (Table 2). α‐Bungarotoxin (10⁻⁶ mol·L⁻¹ at the serosal side), a blocker of of α7, α8 and α9*‐subunits, was ineffective. In contrast, DhβE (10⁻⁵ mol·L⁻¹ at the serosal side), an inhibitor of, for example, α4β2 and α3β2 nicotinic receptors, and atropine (2.5·10⁻⁵ mol·L⁻¹ at the serosal side), which – in concentrations higher than those needed for muscarinic receptor blockade – inhibits, for example, α3β2, α3β4, α4β2 or α4β4 nicotinic receptors (Parker et al., 2003), caused a significant inhibition of the nicotine‐induced pump current (Table 2). Inhibition amounted to about 35% in the case of DhβE and 45% in the case of atropine, that is, inhibition was incomplete.

As the blocking profiles of DhβE and atropine only partially overlap, we tested a combination of both receptor blockers, which might cause a stronger inhibition of the nicotine‐induced pump current. This was indeed the case. When both inhibitors were combined, the nicotine‐induced I_sc was reduced by 60% in comparison with an untreated control group (Table 2). Additional inclusion of strychnine in the blocker ‘cocktail’ did not enhance the inhibition (Table 2), suggesting that, in contrast to atropine‐ and DhβE‐sensitive nicotinic receptors, strychnine‐sensitive nicotinic receptors are probably not involved in the stimulation of the pump current by epithelial nicotinic receptors. The solvents used for the solubilization of some of the blockers, that is, DMSO or ethanol, had no effect on the stimulation of the pump current by nicotine (Table 2).

Despite the fact that all experiments were performed in the presence of TTX, which blocks the propagation of action potentials in neurons, there remains the possibility that nicotine stimulates presynaptic nicotinic receptors (Galligan, 1999). Such a presynaptic effect would be insensitive to TTX. In order to block neurotransmitter release, two snail toxins, conotoxin MVIIC (a blocker of PQ‐ and N‐type voltage‐dependent Ca²⁺ channels) and conotoxin SVIB (a blocker of N‐type voltage‐dependent Ca²⁺ channels), were used. However, when the tissues were pretreated with a combination of both blockers (each in a concentration of 5 · 10⁻⁸ mol·L⁻¹ at the serosal side), the stimulation of the pump current was not inhibited (Table 2), excluding the possibility that nicotine acts via presynaptic nicotinic receptors.

Potentiation of epithelial muscarinic and nicotinic receptor responses during induction of transepithelial Cl⁻ secretion

The colonic epithelium expresses both muscarinic M₁ and M₃ receptors (Haberberger et al., 2006) as well as different subunits of nicotinic receptors (Bader and Diener, 2015). This suggests the possibility that induction of Cl⁻ secretion by acetylcholine or other mixed cholinergic agonists such as carbachol might not only be mediated by stimulation of Ca²⁺‐dependent Cl⁻ secretion via muscarinic receptors involving Ca²⁺‐dependent K⁺ and Cl⁻ channels (Strabel and Diener, 1995, Hennig et al., 2008) but also might be potentiated by the simultaneous stimulation of epithelial nicotinic receptors coupled to the activity of the Na⁺‐K⁺‐pump.

Therefore, we looked for potentiation of the transepithelial Cl⁻ secretion – measured as increase in I_sc in non‐permeabilized epithelia – evoked by simultaneous stimulation of muscarinic and nicotinic receptors with selective agonists for both types of cholinergic receptors. A low concentration of nicotine (10⁻⁶ mol·L⁻¹ at the serosal side) evoked an increase in I_sc of 1.34 ± 0.53 μEq·h⁻¹·cm⁻² (n = 5). A similar increase of 1.35 ± 0.21 μEq·h⁻¹·cm⁻² (n = 7) was induced by the muscarinic agonist pilocarpine in a concentration of 4·10⁻⁶ mol·L⁻¹ (at the serosal side). When both agonists were administered simultaneously, the amplitude of the stimulated I_sc was about fourfold in comparison with the response to any one of these drugs, administered alone (Figure 6). In other words, there was a synergistic action suggesting a potentiation of the effect of muscarinic and nicotinic receptor stimulation on transepithelial anion secretion.

In order to establish the optimal concentration ratio of the nicotinic and the muscarinic agonists for potentiation of the secretory response, the nicotine concentration was fixed to 10⁻⁶ mol·L⁻¹ and the concentration of pilocarpine was varied from 2·10⁻⁶ mol·L⁻¹ to 5·10⁻⁵ mol·L⁻¹ (Figure 6B). Under these conditions, a maximal potentiation was observed for a ratio of nicotine : pilocarpine of 1:10, that is, 10⁻⁶ mol·L⁻¹ nicotine combined with 10⁻⁵ mol·L⁻¹ pilocarpine (Figure 6B). When the concentration of pilocarpine was elevated further to 5·10⁻⁵ mol·L⁻¹, no more overadditive effect was observed, probably because the increase in I_sc evoked by the muscarinic agonist (>10 μEq·h⁻¹·cm⁻²) reached the maximal secretory capacity of the epithelium.

With the ratio of nicotine to pilocarpine fixed to 1:10, in a final series of experiments, the concentration–response curves for low concentrations of nicotine (10⁻⁸ mol·L⁻¹ to 10⁻⁶ mol·L⁻¹ at the serosal side), pilocarpine (10⁻⁷ mol·L⁻¹ to 10⁻⁵ mol·L⁻¹ at the serosal side) and the combination of both agonists were measured. Indeed, there was an overadditive effect, when both types of cholinergic receptors were stimulated synchronously. This effect started at concentrations of 5·10⁻⁷ mol·L⁻¹ nicotine/5·10⁻⁶ mol·L⁻¹ pilocarpine and was clearly visible, when the concentrations were increased to 10⁻⁶ mol·L⁻¹ nicotine/10⁻⁵ mol·L⁻¹ pilocarpine (Figure 7) demonstrating potentiation of muscarinic and nicotinic receptor stimulation in the induction of epithelial secretion.

Figure 7.

Concentration‐dependent effect of nicotine (green), pilocarpine (blue) and the combination of both (red) on transepithelial chloride secretion measured as increase in I_sc (ΔI_sc) in intact epithelia. The agonists were administered at the serosal side. All experiments were performed in the presence of TTX (10⁻⁶ mol·L⁻¹ at the serosal side). After each administration of an agonist (or of the agonist combination), the serosal compartment was washed three times with 5× the chamber volume, before the next concentration was administered. For graphical clarity, two of the three symbols for the same concentration(s) were laterally shifted to the right or the left, respectively, in order to avoid overlay of the data points. Values are given as increase in I_sc (ΔI_sc) and are means ± SEM, n = 5 (nicotine), 6 (pilocarpine) and 8 (combination).

Anion subsitution and transport inhibitor experiments were performed as a final control that the increase in I_sc induced by the combined administration of nicotine (10⁻⁶ mol·L⁻¹ at the serosal side) and pilocarpine (10⁻⁵ mol·L⁻¹ at the serosal side) is indeed carried by a Cl⁻ secretion. When Cl⁻ ions were substituted on both sides of the chamber by the impermeant anion gluconate, the increase in I_sc evoked by the agonist mix only amounted to 0.16 ± 0.04 μEq·h⁻¹·cm⁻² (n = 6), which was significantly smaller compared with a parallely performed control response in Cl⁻‐containing buffer, where an increase in I_sc of 1.42 ± 0.45 μEq·h⁻¹·cm⁻² (n = 9, P < 0.05 vs. response in Cl⁻‐free buffer) was observed. An inhibition of similar amplitude was observed, when the basolateral Na⁺‐K⁺‐2Cl⁻ co‐transporter, the dominant Cl⁻ transporter for uptake of Cl⁻ in the basolateral membrane, was inhibited by bumetanide (10⁻⁴ mol·L⁻¹ at the serosal side). In the presence of this inhibitor, the nicotine/pilocarpine mix induced an increase in I_sc of only 0.29 ± 0.11 μEq·h⁻¹·cm⁻² (n = 6) compared with 1.65 ± 0.45 μEq·h⁻¹·cm⁻² in the absence of bumetanide (n = 6, P < 0.05 vs. response in the presence of bumetanide).

Discussion

Nicotine receptors are not exclusively expressed by electrically excitable cells such as neurons or skeletal muscle fibres, but are found also in other tissues such as epithelia. For example, different subunits of these homo‐ or heteropentameric receptors have been found in epithelia in placenta (Lips et al., 2005), trachea (Kummer et al., 2008) or skin (Wessler and Kirkpatrick, 2008). They are not only involved in the regulation of physiological functions of the epithelium such as control of the production of airway lining fluid in the lung (Hollenhorst et al., 2012) but are thought to play also an important role under pathophysiological conditions such as tumour induction in the lung or in the gastrointestinal tract (Schuller, 2009).

The present results indicate a new mechanism of action by which epithelial nicotinic receptors, recently identified on the molecular and the functional level in rat colonic epithelium (Bader and Diener, 2015), are involved in the control of colonic Cl⁻ secretion. In contrast to stimulation of muscarinic receptors (Strabel and Diener, 1995, Hennig et al., 2008), stimulation of epithelial nicotinic receptors evoked by nicotine (or DMPP) in the presence of TTX was not coupled to the opening of apical Cl⁻ channels (Figure 1) or basolateral K⁺ channels (Figure 3). Both were activated by an increase of the cytosolic Ca²⁺ concentration evoked, for example, by carbachol (Lindqvist et al., 1998). Also, an activation of apical Ca²⁺‐dependent K⁺ channels was not induced by nicotine (data not shown). Such an effect would, however, lead to a negative I_sc as observed in the early phase of the carbachol‐induced I_sc response in rat colon (Schultheiss and Diener, 1997), but might indirectly support anion secretion via maintenance of a negative membrane potential (Cook and Young, 1989). Instead, the stimulation of anion secretion by nicotine takes place at the basolateral membrane (Figure 3) and is caused by the activation of a Na⁺‐dependent, scilliroside‐ and ouabain‐sensitive (Table 1) current. Thus, nicotinic agonists such as nicotine (Figure 4) or DMPP (Figure 5) activate the basolateral Na⁺‐K⁺‐pump.

The function of this ATPase in epithelia and other cells is to establish the central ion concentration gradients between the cytosol and the extracellular medium. The Na⁺ concentration gradient establishes the driving force for the Na⁺‐coupled secondary active transport of other solutes, whereas the K⁺ gradient establishes the basal membrane potential due to diffusion of K⁺ out of the cell via K⁺ channels in the membrane. The Na⁺‐K⁺‐ATPase is composed of a combination of different isoforms of α‐subunits, which contain the Na⁺‐ and K⁺‐binding sites, and β‐subunits, which function as chaperons. In epithelia, including that of rat colon, the dominant form of this enzyme is a α₁β₁ heterodimer (Escoubet et al., 1997). As a facultative component, the regulatory protein FXYD (with seven isoforms known up to now) is described, which seems, however, not to be essential for pump activity (Geering, 2005). The α₁ subunit from rats is known to be about 100× less sensitive to ouabain (Blanco and Mercer, 1998), the prototypical steroid blocking pump activity in most species, which explains the only incomplete inhibition of the nicotine‐induced pump current by ouabain, whereas scilliroside, the most potent blocker of rat Na⁺‐K⁺‐ATPase (Robinson, 1970), nearly abolished it (Table 1).

Different subunits of nicotinic receptors have been found when performing RT‐PCR experiments starting with mRNA isolated from rat colonic crypts (Bader and Diener, 2015). Therefore, inhibitor experiments were designed with subtype‐selective inhibitors (for references to the inhibitors used, see Chavez‐Noriega et al., 1997, Parker et al., 2003, Wonnacott and Barik, 2007) to find out which of the subunit combination(s) that can be thought to form functional nicotinic receptors might mediate the stimulation of the pump current measured in apically permeabilized colonic epithelium. α‐Bungarotoxin (blocker of α7, α8 and α9α10 subunits of nicotinic receptors) was ineffective and also strychnine (blocker of α7, α8 and α9α10 subunits) did not diminish significantly the nicotine‐induced pump current (Table 2). In contrast, DhβE (inhibitor of, e.g. α4β2 and α3β2 nicotinic receptors) and a high concentration of atropine (which inhibits, e.g. α3β2, α3β4, α4β2 or α4β4 nicotinic receptors; Parker et al., 2003) caused a significant inhibition of the nicotine‐induced pump current (Table 2). Inhibition was more effective, when both inhibitors were combined. If one does not assume insufficient concentrations to be responsible for the incomplete inhibition (which is, however, difficult to test because at higher concentrations subtype selectivity becomes less likely), this might indicate that more than one combination of the subunits found in rat colonic epithelium (α2, α4, α5, α6, α7, α10 and β4; Bader and Diener, 2015) participates in the regulation of pump activity.

The mode of action, how these epithelial nicotinic receptors are coupled to Na⁺‐K⁺‐ATPase, is not yet known. Classically, nicotinic receptors are ligand‐gated cation channels that open a non‐selective cation channel as acetylcholine binds to a binding site formed by two neighbouring subunits (α1 to α7 and α9 on one side and α10, β3, β4, γ, δ or ε at the other side; Albuquerque et al., 2009). However, also metabotropic effects of nicotinic receptors have been described (Grando, 2014). One example for a cellular system in which the response evoked by nicotine is independent from cation flux into the cell is the hyperpolarization induced by nicotine at rat septal neurons. This response has been shown to be independent from the influx of extracellular Ca²⁺, but instead involves a G‐protein (Sorenson and Gallagher, 1996). A further example for metabotropic signalling via nicotinic receptors is the neuroprotective effect of nicotine on rat brain, which seems to involve the activation of phosphatidyl inositol‐3‐kinase (PI3K) and of the membrane‐associated tyrosine kinase Src (Kihara et al., 2001). So it remains to be determined whether in rat colonic epithelium nicotine acts via ionotropic receptors, that is, activates cation influx into the cell via ligand‐gated ion channels, or involves metabotropic mechanism(s) of action. Interestingly, stimulation of sugar absorption in porcine jejunum by insulin‐like growth factor 1 is caused by activation of the Na⁺‐K⁺‐pump and is mediated by a PI3 kinase (Alexander and Carey, 2001). Also, the thyroid hormone, triiodothyronine, activates Na⁺‐K⁺‐pump activity in tracheal epithelium via non‐genomic mechanisms involving Src und PI3‐kinase (Bhargava et al., 2007). Thus, there is an overlap of known metabotropic signalling cascades, which can be coupled to nicotinic receptors, with pathways involved in the control of Na⁺‐K⁺‐pump activity. Whether these or other signalling cascades with known effects of the Na⁺‐K⁺‐ATPase such as changes in the phosphorylation level (Poulsen et al., 2010) or changes in redox state (Figtree et al., 2012) or other mechanisms mediate the coupling between nicotinic receptors and Na⁺‐K⁺‐pump activity in the colonic epithelium remains to be determined in future experiments. A further unknown parameter is the localization of nicotinic receptors within the epithelium, for example, the distribution between crypt and surface epithelium. This is, however, a challenging problem due to the lack of specific antibodies (see, e.g. Moser et al., 2007).

Although the intracellular mode of action of epithelial nicotinic receptors is yet unknown, these results challenge the classical view on cholinergic regulation of ion transport, where acetylcholine released from enteric neurons or from colonic epithelium, which produces acetylcholine as a paracrine regulator of epithelial functions, is thought to exert its actions exclusively via epithelial muscarinic M1 and M3 receptors (Haberberger et al., 2006). The current data demonstrate that the simultaneous stimulation of muscarinic and nicotinic receptors potentiates their mutual effect on transepithelial Cl⁻ secretion (Figures 6 and 7). This has, however, to be expected when considering the different ion transporters regulated by both types of cholinergic receptors. Muscarinic M₁ and M₃ receptors couple via Gq_α to phospholipase C (see Hennig et al., 2008) and induce an increase in the cytosolic Ca²⁺ concentration (Lindqvist et al., 1998), which is finally responsible for the activation of Ca²⁺‐dependent K⁺ and Cl⁻ channels (Strabel and Diener, 1995, Hennig et al., 2008). In contrast, stimulation of the electrogenic Na⁺‐K⁺‐ATPase by nicotinic receptors will increase the driving force for K⁺ efflux by elevating the cytosolic K⁺ concentration and hyperpolarize the membrane, which will enhance Cl⁻ currents across apical Cl⁻ channels. Both of these effects enhance the cellular response to muscarinic receptor stimulation. Due to the potentiation of these mechanisms, blockade of either one of them, for example, of muscarinic receptors by atropine, will thus cause a strong inhibition of acetylcholine‐induced anion secretion, which led to the classical model of muscarinic receptor‐mediated epithelial effects of acetylcholine (Zimmerman et al., 1982). However, close inspection of older data reveal an over‐additive inhibitory effect of muscarinic and nicotinic antagonists. For example, both hexamethonium and a low concentration (10⁻⁶ mol·L⁻¹) of atropine, where the drug can be considered to act selectively on muscarinic receptors, inhibit acetylcholine‐induced Cl⁻ secretion by about two‐thirds, that is, show an over‐additive action, and only the combination of both inhibitors suppresses the response to the natural cholinergic agonist (Diener et al., 1989). Similar observations were made recently for the ‘atypical’ choline esters propionyl‐ and butyrylcholine, which – beside acetylcholine – are components of the non‐neuronal cholinergic system of the colonic epithelium (Moreno et al., 2016).

The Na⁺‐K⁺‐ATPase in the basolateral membrane is not only a transport enzyme. Trans‐interactions of the β₁ subunits of neighbouring cells in the region of the desmosomes contribute to the establishment of cellular contacts within the epithelium (Vagin et al., 2012. Furthermore, this molecule is also involved in signal transduction (see Jansson et al., 2015). Under pathophysiological conditions, nicotinic receptors, which are stimulated, for example, by nitrosamine (from processed food), are thought to be involved in tumour induction, as they influence apoptosis, proliferation or migration of cells (Schuller, 2009, Grando, 2014). Thus, it seems to be of importance to investigate the physiological role of epithelial nicotinic receptors and their signalling mechanisms in colon as a rational base for the understanding of their pathophysiological role, for example, in the pathogenesis of colonic cancer, one of the most frequent forms of human cancer.

Author contributions

S.B. and L.L. designed the study, performed experiments, interpreted data and drafted the manuscript. M.D. designed the study, interpreted data and drafted the manuscript. All authors approved the version to be submitted.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Bader, S. , Lottig, L. , and Diener, M. (2017) Stimulation of Na⁺‐K⁺‐pump currents by epithelial nicotinic receptors in rat colon. British Journal of Pharmacology, 174: 880–892. doi: 10.1111/bph.13761.