Tae Sik Sung

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

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 Ca2+‐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.

Intracellular Ca2+ release from endoplasmic reticulum regulates slow wave currents and pacemaker activity of 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 large-amplitude inward currents resulting from anoctamin 1 (ANO1) channels, which are Ca(2+)-activated Cl(-) channels. We investigated the hypothesis that the Ca(2+) responsible for the stochastic activation of ANO1 channels during spontaneous transient inward currents (STICs) and synchronized activation of ANO1 channels during slow wave currents comes from intracellular Ca(2+) stores. ICC, obtained from the small intestine of Kit(+/copGFP) mice, were studied under voltage and current clamp to determine the effects of blocking Ca(2+) uptake into stores and release of Ca(2+) via inositol 1,4,5-trisphosphate (IP3)-dependent and ryanodine-sensitive channels. Cyclocpiazonic acid, thapsigargin, 2-APB, and xestospongin C inhibited STICs and slow wave currents. Ryanodine and tetracaine also inhibited STICs and slow wave currents. Store-active compounds had no direct effects on ANO1 channels expressed in human embryonic kidney-293 cells. Under current clamp, store-active drugs caused significant depolarization of ICC and reduced spontaneous transient depolarizations (STDs). After block of ryanodine receptors with ryanodine and tetracaine, repolarization did not restore STDs. ANO1 expressed in ICC has limited access to cytoplasmic Ca(2+) concentration, suggesting that pacemaker activity depends on Ca(2+) dynamics in restricted microdomains. Our data from studies of isolated ICC differ somewhat from studies on intact muscles and suggest that release of Ca(2+) from both IP3 and ryanodine receptors is important in generating pacemaker activity in ICC.

Influence of intracellular Ca2+ and alternative splicing on the pharmacological profile of ANO1 channels

Anoctamin-1 (ANO1) is a Ca(2+)-activated Cl(-) channel expressed in many types of cells. Splice variants of ANO1 have been shown to influence the biophysical properties of conductance. It has been suggested that several new antagonists of ANO1 with relatively high affinity and selectivity might be useful for experimental and, potentially, therapeutic purposes. We investigated the effects of intracellular Ca(2+) concentration ([Ca(2+)]i) at 100-1,000 nM, a concentration range that might be achieved in cells during physiological activation of ANO1 channels, on blockade of ANO1 channels expressed in HEK-293 cells. Whole cell and excised patch configurations of the patch-clamp technique were used to perform tests on a variety of naturally occurring splice variants of ANO1. Blockade of ANO1 currents with aminophenylthiazole (T16Ainh-A01) was highly dependent on [Ca(2+)]i Increasing [Ca(2+)]i reduced the potency of this blocker. Similar Ca(2+)-dependent effects were also observed with benzbromarone. Experiments on excised, inside-out patches showed that the diminished potency of the blockers caused by intracellular Ca(2+) might involve a competitive interaction for a common binding site or repulsion of the blocking drugs by electrostatic forces at the cytoplasmic surface of the channels. The degree of interaction between the channel blockers and [Ca(2+)]i depends on the splice variant expressed. These experiments demonstrate that the efficacy of ANO1 antagonists depends on [Ca(2+)]i, suggesting a need for caution when ANO1 blockers are used to determine the role of ANO1 in physiological functions and in their use as therapeutic agents.

Clustering of Ca2+ transients in interstitial cells of Cajal defines slow wave duration

Interstitial cells of Cajal (ICC) in the myenteric plexus region (ICC-MY) of the small intestine are pacemakers that generate rhythmic depolarizations known as slow waves. Slow waves depend on activation of Ca2+-activated Cl− channels (ANO1) in ICC, propagate actively within networks of ICC-MY, and conduct to smooth muscle cells where they generate action potentials and phasic contractions. Thus, mechanisms of Ca2+ regulation in ICC are fundamental to the motor patterns of the bowel. Here, we characterize the nature of Ca2+ transients in ICC-MY within intact muscles, using mice expressing a genetically encoded Ca2+ sensor, GCaMP3, in ICC. Ca2+ transients in ICC-MY display a complex firing pattern caused by localized Ca2+ release events arising from multiple sites in cell somata and processes. Ca2+ transients are clustered within the time course of slow waves but fire asynchronously during these clusters. The durations of Ca2+ transient clusters (CTCs) correspond to slow wave durations (plateau phase). Simultaneous imaging and intracellular electrical recordings revealed that the upstroke depolarization of slow waves precedes clusters of Ca2+ transients. Summation of CTCs results in relatively uniform Ca2+ responses from one slow wave to another. These Ca2+ transients are caused by Ca2+ release from intracellular stores and depend on ryanodine receptors as well as amplification from IP3 receptors. Reduced extracellular Ca2+ concentrations and T-type Ca2+ channel blockers decreased the number of firing sites and firing probability of Ca2+ transients. In summary, the fundamental electrical events of small intestinal muscles generated by ICC-MY depend on asynchronous firing of Ca2+ transients from multiple intracellular release sites. These events are organized into clusters by Ca2+ influx through T-type Ca2+ channels to sustain activation of ANO1 channels and generate the plateau phase of slow waves.

Na+-K+-Cl− cotransporter (NKCC) maintains the chloride gradient to sustain pacemaker activity in 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 significant, as there is high current density during slow-wave currents and slow waves are of sufficient magnitude to depolarize the syncytium of smooth muscle cells and PDGFRα+ cells to which they are electrically coupled. We investigated how the driving force for Cl- current is maintained in ICC. We found robust expression of Slc12a2 (which encodes an Na+-K+-Cl- cotransporter, NKCC1) and immunohistochemical confirmation that NKCC1 is expressed in ICC. With the use of the gramicidin permeabilized-patch technique, which is reported to not disturb [Cl-]i, the reversal potential for spontaneous transient inward currents (ESTICs) was -10.5 mV. This value corresponds to the peak of slow waves when they are recorded directly from ICC in situ. Inhibition of NKCC1 with bumetanide shifted ESTICs to more negative potentials within a few minutes and reduced pacemaker activity. Bumetanide had no direct effects on ANO1 or CaV3.2 channels expressed in HEK293 cells or L-type Ca2+ currents. Reducing extracellular Cl- to 10 mM shifted ESTICs to positive potentials as predicted by the Nernst equation. The relatively rapid shift in ESTICs when NKCC1 was blocked suggests that significant changes in the transmembrane Cl- gradient occur during the slow-wave cycle, possibly within microdomains formed between endoplasmic reticulum and the plasma membrane in ICC. Recovery of Cl- via NKCC1 might have additional consequences on shaping the waveforms of slow waves via Na+ entry into microdomains.

Protease-activated receptors modulate excitability of murine colonic smooth muscles by differential effects on interstitial cells.

Activation of protease‐activated receptors (PAR) regulates gastrointestinal (GI) motility but little is known about the cells and mechanisms in GI muscles responsible for PAR responses. Using mouse cells, we found high levels of F2r and F2rl1 PAR‐encoding genes expressed in purified platelet‐derived growth factor α‐positive (PDGFRα+) cells in comparison to other cells in colonic muscles. PAR1 and PAR2 agonists caused transient hyperpolarization and relaxation of colonic muscles, with relaxation responses followed by excitation. The inhibitory phase was inhibited by apamin and mediated by activation of small conductance calcium‐activated potassium channels in PDGFRα+ cells. The excitatory response resulted largely from activation of a chloride conductance in interstitial cells of Cajal; small amplitude inward currents were generated in smooth muscle cells by PAR activation, but these responses were too small to be resolved in intact muscles. PAR receptor responses are integrated responses generated by cells of the smooth muscle, interstitial cells of Cajal and PDGFRα+ cells (SIP syncytium).

The cells and conductance mediating cholinergic neurotransmission in the murine proximal stomach

Enteric neurotransmission is essential for gastrointestinal (GI) motility, although the cells and conductances responsible for post‐junctional responses are controversial. The calcium‐activated chloride conductance (CaCC), anoctamin‐1 (Ano1), was expressed by intramuscular interstitial cells of Cajal (ICC‐IM) in proximal stomach and not resolved in smooth muscle cells (SMCs). Cholinergic nerve fibres were closely apposed to ICC‐IM. Conductances activated by cholinergic stimulation in isolated ICC‐IM and SMCs were determined. A CaCC was activated by carbachol in ICC‐IM and a non‐selective cation conductance in SMCs. Responses to cholinergic nerve stimulation were studied. Excitatory junction potentials (EJPs) and mechanical responses were evoked in wild‐type mice but absent or greatly reduced with knockout/down of Ano1. Drugs that block Ano1 inhibited the conductance activated by carbachol in ICC‐IM and EJPs and mechanical responses in tissues. The data of the present study suggest that electrical and mechanical responses to cholinergic nerve stimulation are mediated by Ano1 expressed in ICC‐IM and not SMCs.

Correction: Clustering of Ca 2+ transients in interstitial cells of Cajal defines slow wave duration

Volume 149, No. 7, July, 2017. [https://doi.org/10.1085/jgp.201711771][1] The authors regret that in the original version of their paper, some of the values given in the KRB solution were incorrect. The corrected subsection of the Materials and methods appears below in its entirety: ### Drugs and

What is the next step for gastric atypical epithelium on histological findings of endoscopic forceps biopsy

BACKGROUND Gastric atypical epithelium on endoscopic biopsy is borderline lesions between benign and malignant. Definitive management of this lesion remains debatable. AIMS We aimed to analyze the final histological diagnosis for atypical epithelium on endoscopic biopsy and to examine the discrepancy rate between the final histological diagnosis and the initial endoscopic assessment. METHODS This retrospective study finally enrolled 24 cases proven atypical epithelium on initial histology of an endoscopic biopsy. Of 24 cases, endoscopic submucosal dissection (n = 22), operation (n = 1) and follow-up biopsy without endoscopic submucosal dissection (n = 1) were performed. RESULTS Of the 24 cases, early gastric cancer (n = 15, 62%) and adenoma (n = 7, 30%) lesions were finally diagnosed in 22 cases. Age, sex, endoscopic results and number of biopsy did not significantly influence the result of final outcome. Between the initial endoscopic assessment and the final histological diagnosis, 12 cases (50%) showed a concordant diagnosis, but eight (33%) and four cases (17%) showed upgraded and downgraded diagnoses, respectively. CONCLUSIONS Of atypical epithelium cases, the rate of malignant and premalignant lesions was 92% and it was difficult to distinguish between malignant and benign lesions using the initial endoscopic findings. Therefore, endoscopic submucosal dissection can be considered in patients with atypical epithelium on endoscopic biopsy.

SOCE mediated by STIM and Orai is essential for pacemaker activity in the interstitial cells of Cajal in the gastrointestinal tract

Refilling ER Ca2+ stores through SOCE enables the interstitial cells of Cajal to trigger pacemaker activity in the gut. Keeping the gut moving The gut contracts periodically to mix and propel its contents. These rhythmic contractions are driven by the pacemaker activity of the interstitial cells of Cajal in the gut, which generate electrical slow waves that require Ca2+ release from the endoplasmic reticulum (ER). Zheng et al. showed that ER Ca2+ stores in the interstitial cells of Cajal were refilled by store-operated Ca2+ entry (SOCE), a process that was mediated by Orai Ca2+ channels and the STIM family of Ca2+ sensors. Some gastrointestinal motility disorders have been linked to defects in the activity of the interstitial cells of Cajal, which may be due to dysfunctional SOCE. Electrical pacemaker activity generates phasic contractions and motility patterns such as segmentation and peristalsis in the gastrointestinal tract. Pacemaker currents are generated in interstitial cells of Cajal (ICC), which release Ca2+ from intracellular stores that stimulates Ca2+-activated Cl− channels (CaCCs) in the plasma membrane. Thus, Ca2+ stores must be maintained to sustain pacemaker activity. Store-operated Ca2+ entry (SOCE) facilitates the refilling of Ca2+ stores by a mechanism dependent upon interactions between STIM and Orai proteins. We investigated the role of SOCE in ICC pacemaker activity. Reintroduction of extracellular Ca2+ in store-depleted ICC resulted in CaCC activation. Blocking CaCCs revealed an inwardly rectifying current with properties of a Ca2+ release–activated current (ICRAC). An inhibitory peptide that interfered with the STIM-Orai interaction blocked ICRAC in HEK 293 cells expressing STIM1 and Orai1 and blocked spontaneous transient inward currents (STICs) and slow wave currents in ICC. STICs, which are fundamental pacemaker events in ICC, were blocked by an Orai antagonist. Imaging of Ca2+ transients linked to pacemaker activity in ICC in intact muscles showed that the Orai antagonist blocked Ca2+ transients in ICC. These data suggest that Ca2+ recovery through STIM-Orai interactions is necessary to maintain ICC pacemaker activity.