The importance of the gut-brain axis in maintaining homeostasis has long been appreciated. However, the past 15 yr have seen the emergence of the microbiota (the trillions of microorganisms within ...

The Microbiota-Gut-Brain Axis

Let X(t) be the number of tumor cells at time t, and Pr{X(t) = n} = pn(t) is the density of X. A “birth”, i.e., an increase of one of the total population of cancer cells, can occur either by mutation of a normal cell caused by the action of the carcinogen, consisting of randomly (Poisson) distributed hits, or by reproduction of existing cancer cells. A death of a tumor cell occurs as an additive combination of non-immunological and immunological elements. Once a tumor is initiated by carcinogenic action, it undergoes a birth and death process with infinitesimal birth rate linear and infinitesimal death rate composed of a linear and a nonlinear term, the former due to non-immunological deaths, the latter to immunological feedback. The death rate per tumor cell due to immunological response is proportional to the total number of antigen-producing (tumor) cells; thus, the total death rate is quadratic. Although this assumes a very simple mechanism for the action of immunological feedback, it is nevertheless a first step.

The Mathematical Model

TMEM16 proteins, also known as anoctamins, are involved in a variety of functions that include ion transport, phospholipid scrambling, and regulation of other membrane proteins. The first two membe...

Structure and Function of TMEM16 Proteins (Anoctamins)

Specialized cells, referred to as interstitial cells of Cajal (ICC), generate and actively propagate the spontaneous electrical activity known as slow waves in the gastrointestinal (GI) tract (Ward et al. 1994, Huizinga et al. 1995; Sanders, 1996). Slow waves time the phasic contractions of GI muscles and provide the underlying organization of excitability for gastric peristalsis and intestinal segmentation. ICC are also interposed between nerve terminals and smooth muscle cells and serve as sites of post-junctional transduction of responses to enteric motor neurotransmitters (see Burns et al. 1996; Ward et al. 2000a). There are at least two classes of ICC, those intermingled within muscle bundles of the circular (CM) and longitudinal muscle (LM) layers (ICC-IM), and those arranged in a dense network between the CM and LM (ICC-MY), at the submucosal surface of the CM (ICC-SM) and in the septa between bundles of smooth muscle cells (ICC-SEP). There have been many studies regarding the mechanisms of pacemaker activity and slow wave propagation, but the precise mechanisms of this behaviour remain controversial.

Studies of pacemaker activity in intact muscle strips and bundles have suggested the involvement of a Ca2+-dependent inward current because activity was reduced when the muscles were treated with membrane-permeable Ca2+ buffers (Edwards et al. 1999; Hirst et al. 2002; Kito & Suzuki, 2003). The Ca2+-dependent conductance has been thought to be a Cl− conductance, since a variety of Cl− channel blocking drugs reduced pacemaker activity in guinea-pig and murine muscles (see Hirst et al. 2002; Kito et al. 2002a; Kito & Suzuki, 2003). Similar conclusions were reached from studies of slow waves recorded directly from ICC-MY of the small intestine (e.g. Kito & Suzuki, 2003). In contrast, studies of isolated and cultured ICC have suggested that spontaneous activity is generated by activation of a non-selective cation conductance (Ward et al. 2000; Koh et al. 2002; Sanders et al. 2006), and the putative conductance was found to be inhibited by Ca2+ (Koh et al. 2002). Thus, pacemaker current may be initiated by a transient reduction in [Ca2+]i in a sub-compartment under the plasma membrane containing the non-selective cation conductance (Sanders et al. 2006). No Ca2+-activated inward currents were observed in cultured ICC, and the non-selective cation channels activated by reduced Ca2+ were inhibited by niflumic acid (Koh et al. 2002). Thus, use of Cl− channel antagonists does not necessarily indicate a role for Ca2+- activated Cl− channels in pacemaker activity.

A microarray genetic screen recently revealed that Tmem16a is expressed at far greater levels in ICC than in the rest of the muscularis (Chen et al. 2007). Tmem16a encodes ANO1, a Ca2+-activated Cl− channel (Caputo et al. 2008; Schroeder et al. 2008; Yang et al. 2008), and immunohistochemical studies have documented expression of ANO1 (also known as DOG1) protein by ICC (Espinosa et al. 2008; Gomez-Pinilla et al. 2009). Taken together these data suggest the hypothesis that expression and function of these channels may be important in pacemaker activity in the GI tract. Therefore, we have characterized expression of Tmem16a transcripts and ANO1 protein in the tunica muscularis of mouse, monkey (Macaca fascicularis) and human GI tracts using RT-PCR, amplicon sequencing and immunohistochemical techniques. We also evaluated the electrical activity of murine gastric and small intestine muscles, and tested the effects of Cl− channel-blocking drugs. Finally, we tested whether slow wave activity is affected in mice homozygous with null Tmem16a alleles (Tmem16atm1Bdh/tm1Bdh, see Rock et al. 2008). Our data show ubiquitous expression of ANO1 in ICC throughout the GI tract and inhibitory effects of Cl− channel blocking drugs on slow waves. Tmem16a−/− animals failed to generate slow waves, and pacemaker activity did not develop in organ culture after birth, as occurs in wildtype muscles. Together with voltage-clamp studies of isolated ICC (Zhu et al. 2009), our findings strongly support a role for ANO1 in the generation of slow wave currents of GI ICC and electrical slow waves in intact muscles. The model of pacemaker activity deduced from previous studies of cultured ICC (e.g. as detailed in Sanders et al. 2006) will require reconsideration in light of these new findings.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.2009.176198

Expression of anoctamin 1/TMEM16A by interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles

Smooth muscles are complex tissues containing a variety of cells in addition to muscle cells. Interstitial cells of mesenchymal origin interact with and form electrical connectivity with smooth muscle cells in many organs, and these cells provide important regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and PDGFRα+ cells have been described, in detail, and represent distinct classes of cells with unique ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled to ICC and PDGFRα+ cells, forming an integrated unit called the SIP syncytium. SIP cells express a variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity, propagation pathways for slow waves, transduction of inputs from motor neurons, and mechanosensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial cells are also found in a variety of other smooth muscles; however, in most cases, the physiological and pathophysiological roles for these cells have not been clearly defined. This review describes structural, functional, and molecular features of interstitial cells and discusses their contributions in determining the behaviors of smooth muscle tissues.

https://www.physiology.org/doi/pdf/10.1152/physrev.00037.2013

Interstitial Cells: Regulators of Smooth Muscle Function

Interstitial cells of Cajal (ICC) are unique cells that generate electrical pacemaker activity in gastrointestinal (GI) muscles. Many previous studies have attempted to characterize the conductances responsible for pacemaker current and slow waves in the GI tract, but the precise mechanism of electrical rhythmicity is still debated. We used a new transgenic mouse with a bright green fluorescent protein (copGFP) constitutively expressed in ICC to facilitate study of these cells in mixed cell dispersions. We found that ICC express a specialized ‘slow wave’ current. Reversal of tail current analysis showed this current was due to a Cl− selective conductance. ICC express ANO1, a Ca2+-activated Cl− channel. Slow wave currents are not voltage dependent, but a secondary voltage-dependent process underlies activation of these currents. Removal of extracellular Ca2+, replacement of Ca2+ with Ba2+, or extracellular Ni2+ (30 μm) blocked the slow wave current. Single Ca2+-activated Cl− channels with a unitary conductance of 7.8 pS were resolved in excised patches of ICC. These are similar in conductance to ANO1 channels (8 pS) expressed in HEK293 cells. Slow wave current was blocked in a concentration-dependent manner by niflumic acid (IC50= 4.8 μm). Slow wave currents are associated with transient depolarizations of ICC in current clamp, and these events were blocked by niflumic acid. These findings demonstrate a role for a Ca2+-activated Cl− conductance in slow wave current in ICC and are consistent with the idea that ANO1 participates in pacemaker activity.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.2009.176206

A Ca2+-activated Cl− conductance in interstitial cells of Cajal linked to slow wave currents and pacemaker activity

Interstitial cells of Cajal (ICC) generate electrical pacemaker activity in gastrointestinal smooth muscles. We investigated whether Tmem16a, which encodes anoctamin 1 (ANO1), a Ca(2+)-activated Cl(-) channel, might be involved in pacemaker activity in ICC. The Tmem16a transcripts and ANO1 were expressed robustly in GI muscles, specifically in ICC in murine, non-human primate (Macaca fascicularis) and human GI tracts. Splice variants of Tmem16a, as well as other paralogues of the Tmem16 family, were expressed in gastrointestinal muscles. Calcium-activated Cl(-) channel blocking drugs, niflumic acid and DIDS blocked slow waves in intact muscles of mouse, primate and human small intestine and stomach. Slow waves failed to develop in Tmem16a knock-out mice (Tmem16a(tm1Bdh/tm1Bdh)). The pacemaker mechanism was investigated in isolated ICC from transgenic mice with constitutive expression of copepod super green fluorescent protein (copGFP). Depolarization of ICC activated inward currents due to a Cl(-)-selective conductance. Removal of extracellular Ca(2+), replacement of Ca(2+) with Ba(2+), or extracellular Ni(2+) (30 μM) blocked the inward current. Single Ca(2+)-activated Cl(-) channels with a unitary conductance of 7.8 pS were resolved in excised patches from ICC. The inward current was blocked in a concentration-dependent manner by niflumic acid (IC(50) = 4.8 μM). The role of ANO1 in cholinergic responses in ICC was also investigated. Carbachol activated Ca(2+)-activated Cl(-) currents in ICC, and responses to cholinergic nerve stimulation were blocked by niflumic acid in intact muscles. Anoctamin 1 is a prominent conductance in ICC, and these channels appear to be involved in pacemaker activity and in responses to enteric excitatory neurotransmitters.

/pdf/anoctamins-and-gastrointestinal-smooth-muscle-excitability-4ppvkmk0o4.pdf

Anoctamins and gastrointestinal smooth muscle excitability

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 nerve stimulation.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.2011.211094

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

Interstitial cells of Cajal (ICC) provide pacemaker activity in gastrointestinal muscles that underlies segmental and peristaltic contractions. ICC generate electrical slow waves that are due to la...

https://www.physiology.org/doi/pdf/10.1152/ajpcell.00360.2014

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

Interstitial cells of Cajal (ICC) generate electrical slow waves by coordinated openings of ANO1 channels, a Ca2+-activated Cl− (CaCC) conductance. Efflux of Cl− during slow waves must be significa...

https://www.physiology.org/doi/pdf/10.1152/ajpgi.00277.2016

Mei Hong Zhu

Papers

A Ca2+-activated Cl− conductance in interstitial cells of Cajal linked to slow wave currents and pacemaker activity

Anoctamins and gastrointestinal smooth muscle excitability

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

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

Na+-K+-Cl- cotransporter (NKCC) maintains the chloride gradient to sustain pacemaker activity in interstitial cells of Cajal