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.

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.

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.

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.

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.

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.

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.

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).