Evidence for metabotropic function of epithelial nicotinic cholinergic receptors in rat colon.

BACKGROUND AND PURPOSE: ACh exerts its actions via nicotinic (nAChR) and muscarinic receptors. In the peripheral nervous system, ionotropic nAChR mediate responses in excitable cells. However, recent studies demonstrate the expression of nAChR in the colonic epithelium, which are coupled to an induction of Cl- secretion via activation of the Na+ -K+ -pump. EXPERIMENTAL APPROACH: In order to find out whether these epithelial nAChR function as ionotropic receptors, intracellular microelectrode and imaging experiments were performed in isolated crypts from rat colon. Apically permeabilized epithelia were used to measure pump current across the basolateral membrane. KEY RESULTS: Imaging experiments with the Na+ -sensitive dye SBFI revealed that nicotine induced a decrease in the cytosolic Na+ concentration concomitant with a fall in the cytosolic Ca2+ concentration in about 50% of the cells. as shown in fura-2 experiments. Nicotine hyperpolarized the membrane by 6.4 ± 2.1 mV. These observations contradict the assumption that epithelial nAChR function as ligand-gated non-selective cation channels. The decrease in the cytosolic Na+ concentration was strongly delayed, when the Na+ -K+ -pump was inhibited by scilliroside. Ussing chamber experiments revealed a strong dependence of the nicotine-induced pump current on the presence of Ca2+ , and chelation of cytosolic Ca2+ with BAPTA prevented the fall in the cytosolic Na+ concentration in SBFI-loaded crypts. Inhibition of PKC with GF 109203X or Goe 6983 significantly reduced the nicotine-induced pump current. CONCLUSIONS AND IMPLICATIONS: These results suggest that epithelial nAChR activate the Na+ -K+ -pump via a PKC dependent on a sufficient cytosolic Ca2+ concentration.

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

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.