Muscarinic activation of Ca²⁺‐activated Cl⁻ current in interstitial cells of Cajal

Abstract

Non-technical summary Interstitial cells of Cajal (ICC) are tightly associated with excitatory and inhibitory motor neurons in the gastrointestinal tract, and these cells are also connected electrically to smooth muscle cells. We have suggested that ICC participate in responses to neurotransmitters released from neurons that drive motility and help move nutrients and wastes through the gut. We studied responses of isolated ICC to cholinergic neurotransmitter and found that a Ca 2+ -activated Cl − current is activated in ICC in response to cholinergic stimulation. Such a current would result in depolarization that could be conducted to surrounding smooth muscle cells by the electrical connections. Exciting ICC would cause generalized excitation of the smooth muscle tissue. A different conductance is activated in smooth muscle cells by cholinergic stimulation. We tested drugs that blocked the Cl − current in ICC and found that responses to nerve stimulation in intact intestinal muscles were blocked by these drugs. This suggests that ICC mediate electrical responses to cholinergic nerve stimulation. In some human gastrointestinal motility disorders, ICC are damaged or lost. If these cells provide responses to neurotransmitters, this might provide an explanation for motor dysfunction in the gut.Abstract Interstitial cells of Cajal (ICC) provide pacemaker activity and functional bridges between enteric motor nerve terminals and gastrointestinal smooth muscle cells. The ionic conductance(s) in ICC that are activated by excitatory neural inputs are unknown. Transgenic mice (Kit copGFP/+ ) with constitutive expression of a bright green fluorescent protein were used to investigate cellular responses of ICC to cholinergic stimulation. ICC displayed spontaneous transient inward currents (STICs) under voltage clamp that corresponded to spontaneous transient depolarizations (STDs) under current clamp. STICs reversed at 0 mV when E Cl = 0 mV and at -40 mV when E Cl was -40 mV, suggesting the STICs were due to a chloride conductance. Carbachol (CCh, 100 nM and 1 μM) induced a sustained inward current (depolarization in current clamp) and increased the amplitude and frequency of STICs and STDs. CCh responses were blocked by atropine (10 μM) or 4-DAMP (100 nM), an M 3 receptor antagonist. STDs were blocked by niflumic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (both 100 μM), and CCh had no effect in the presence of these drugs. The responses of intact circular muscles to CCh and stimulation of intrinsic excitatory nerves by electrical field stimulation (EFS) were also compared. CCh (1 μM) caused atropine-sensitive depolarization and increased the maximum depolarization of slow waves. Similar atropine-sensitive responses were elicited by stimulation of intrinsic excitatory neurons. Niflumic acid (100 μM) blocked responses to EFS but had minor effect on responses to exogenous CCh. These data suggest that different ionic conductances are responsible for electrical responses elicited by bath-applied CCh and cholinergic ne...

The Significance of Interstitial Cells in Neurogastroenterology

“…A more recent study showed that Ca 2+ -activated chloride channels are activated in ICC in response to cholinergic neurotransmission. 205 The channel involved is likely to be ANO1 expressed exclusively in ICC in the GI tract. 67,106,107,131 Additionally, inhibition of a basally active Ca 2+ -activated chloride channel has been suggested to account for part of the response to nitrergic stimulation.…”

Ano1 is a better marker than c-Kit for transcript analysis of single interstitial cells of Cajal in culture

“…To date, only one transcriptomic analysis of ICC has been achieved after enrichment and purification of cell samples through fluorescence-activated cell sorting [9], but the implementation and validation of such a method requires specialized equipment and considerable economic investment. With the availability of transgenic mice with copGFP-expressing ICC [10], primary cultures have been used to improve identification in electrophysiological or immunohistochemical analyses [11,12] but not in single cell characterization. One alternative for the molecular analysis of ICC is the single-cell RT-PCR technique, which allows the collection of individual cells from a mixed culture [13].…”

Intracellular Ca2+ release from endoplasmic reticulum regulates slow wave currents and pacemaker activity of interstitial cells of Cajal

“…STICs generate depolarization, activate Ca 2ϩ entry, and synchronize the openings of channels responsible for STICs, generating slow wave currents. STICs and slow wave currents are due to the activation of Ca 2ϩ -activated Cl Ϫ channels (CaCC) encoded by Ano1 (11,14,53,54). While the mechanism and ionic conductance responsible for STICs and slow wave currents have been described, the mechanisms responsible for delivering Ca 2ϩ to ANO1 channels are not fully understood and have not been studied directly at the level of single ICC.…”

“…For the experiments in this study, we used cells from Kit ϩ/copGFP mice, which have constitutive expression of copGFP in ICC, to investigate the effects of Ca 2ϩ store-active drugs on STICs and slow wave currents (53,54). We studied the effects of blocking Ca 2ϩ uptake into stores and of blocking release of Ca 2ϩ from IP 3 -dependent and ryanodine-sensitive channels to test directly the hypothesis that Ca 2ϩ release from the endoplasmic reticulum (ER) is fundamental to pacemaker activity in ICC.…”