Sang Don Koh

Spontaneous electrical rhythmicity in cultured interstitial cells of Cajal from the murine small intestine

1 Interstitial cells of Cajal (ICC) are pacemaker cells in the small bowel, and therefore this cell type must express the mechanism responsible for slow wave activity. Isolated ICC were cultured for 1–3 days from the murine small intestine and identified with c‐Kit‐like immunoreactivity (c‐Kit‐LI). 2 Electrical recordings were obtained from cultured ICC with the whole‐cell patch clamp technique. ICC were rhythmically active, producing regular slow wave depolarizations with waveforms and properties similar to slow waves in intact tissues. 3 Spontaneous activity of c‐Kit‐LI cells was inhibited by reduced extracellular Na+, gadolinium, and reduced extracellular Ca2+. The activity was not affected by nisoldipine. Voltage clamp studies showed rhythmic inward currents that were probably responsible for the slow wave activity. The current‐voltage relationship showed that the spontaneous currents reversed at about +17 mV. These observations are consistent with the involvement of a non‐selective cation current in the generation of slow waves, but do not rule out contributions from other conductances or transporters. 4 A Ba2+‐sensitive inwardly rectifying K+ current in c‐Kit‐LI cells that may be involved in slow wave repolarization and maintenance of a negative potential between slow waves was also found. Similar pharmacology was observed in studies of intact murine intestinal muscles. 5 Cultured ICC may be a useful model for studying the properties and pharmacology of some of the ionic conductances involved in spontaneous rhythmicity in the gastrointestinal tract.

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

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.

Interstitial Cells: Regulators of Smooth Muscle Function

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.

A Ca2+‐inhibited non‐selective cation conductance contributes to pacemaker currents in mouse interstitial cell of Cajal

Interstitial cells of Cajal (ICC) provide pacemaker activity in some smooth muscles. The nature of the pacemaker conductance is unclear, but studies suggest that pacemaker activity is due to a voltage‐independent, Ca2+‐regulated, non‐selective cation conductance. We investigated Ca2+‐regulated conductances in murine intestinal ICC and found that reducing cytoplasmic Ca2+ activates whole‐cell inward currents and single‐channel currents. Both the whole‐cell currents and single‐channel currents reversed at 0 mV when the equilibrium potentials of all ions present were far from 0 mV. Recordings from on‐cell patches revealed oscillations in unitary currents at the frequency of pacemaker currents in ICC. Voltage‐clamping cells to −60 mV did not change the oscillatory activity of channels in on‐cell patches. Depolarizing cells with high external K+ caused loss of resolvable single‐channel currents, but the oscillatory single‐channel currents were restored when the patches were stepped to negative potentials. Unitary currents were also resolved in excised patches. The single‐channel conductance was 13 pS, and currents reversed at 0 mV. The channels responsible were strongly activated by 10−7m Ca2+, and 10−6m Ca2+ reduced activity. The 13 pS channels were strongly activated by the calmodulin inhibitors calmidazolium and W‐7 in on‐cell and excised patches. Calmidazolium and W‐7 also activated a persistent inward current under whole‐cell conditions. Murine ICC express Ca2+‐inhibited, non‐selective cation channels that are periodically activated at the same frequency as pacemaker currents. This conductance may contribute to the pacemaker current and generation of electrical slow waves in GI muscles.

A functional role for the 'fibroblast-like cells' in gastrointestinal smooth muscles.

Smooth muscles, as in the gastrointestinal tract, are composed of several types of cells. Gastrointestinal muscles contain smooth muscle cells, enteric neurons, glial cells, immune cells, and various classes of interstitial cells. One type of interstitial cell, referred to as ‘fibroblast‐like cells’ by morphologists, are common, but their function is unknown. These cells are found near the terminals of enteric motor neurons, suggesting they could have a role in generating neural responses that help control gastrointestinal movements. We used a novel mouse with bright green fluorescent protein expressed specifically in the fibroblast‐like cells to help us identify these cells in the mixture of cells obtained when whole muscles are dispersed with enzymes. We isolated these cells and found they respond to a major class of inhibitory neurotransmitters – purines. We characterized these responses, and our results provide a new hypothesis about the role of fibroblast‐like cells in smooth muscle tissues.

Voltage-dependent inward currents of interstitial cells of Cajal from murine colon and small intestine

Electrical slow waves in gastrointestinal (GI) muscles are generated by pacemaker cells, known as interstitial cells of Cajal (ICC). The pacemaker conductance is regulated by periodic release of Ca2+ from inositol 1,4,5‐trisphosphate (IP3) receptor‐operated stores, but little is known about how slow waves are actively propagated. We investigated voltage‐dependent Ca2+ currents in cultured ICC from the murine colon and small intestine. ICC, identified by kit immunohistochemistry, were spontaneously active under current clamp and generated transient inward (pacemaker) currents under voltage clamp. Depolarization activated inward currents due to entry of Ca2+. Nicardipine (1 μM) blocked only half of the voltage‐dependent inward current. After nicardipine, there was a shift in the potential at which peak current was obtained (‐15 mV), and negative shifts in the voltage dependence of activation and inactivation of the remaining voltage‐dependent inward current. The current that was resistant to dihydropyridine (IVDDR) displayed kinetics, ion selectivity and pharmacology that differed from dihydropyridine‐sensitive Ca2+ currents. IVDDR was increased by elevating extracellular Ca2+ from 2 to 10 mm, and this caused a +30 mV shift in reversal potential. IVDDR was blocked by Ni2+ (100 μM) or mebefradil (1 μM) but was not affected by blockers of N‐, P‐ or Q‐type Ca2+ channels. Equimolar replacement of Ca2+ with Ba2+ reduced IVDDR without effects on inactivation kinetics. BayK8644 had significantly less effect on IVDDR than on IVDIC. In summary, two components of inward Ca2+ current were resolved in ICC of murine small intestine and colon. Since slow waves persist in the presence of dihydropyridines, the dyhydropyridine‐resistant component of inward current may contribute to slow wave propagation.

Basal Activation of ATP-Sensitive Potassium Channels in Murine Colonic Smooth Muscle Cell

The function and molecular expression of ATP-sensitive potassium (KATP) channels in murine colonic smooth muscle was investigated by intracellular electrical recording from intact muscles, patch-clamp techniques on isolated smooth muscle myocytes, and reverse transcription polymerase chain reaction (RT-PCR) on isolated cells. Lemakalim (1 microM) caused hyperpolarization of intact muscles (17. 2 +/- 3 mV). The hyperpolarization was blocked by glibenclamide (1-10 microM). Addition of glibenclamide (10 microM) alone resulted in membrane depolarization (9.3 +/- 1.7 mV). Lemakalim induced an outward current of 15 +/- 3 pA in isolated myocytes bathed in 5 mM external K+ solution. Application of lemakalim to cells in symmetrical K+ solutions (140/140 mM) resulted in a 97 +/- 5 pA inward current. Both currents were blocked by glibenclamide (1 microM). Pinacidil (1 microM) also activated an inwardly rectifying current that was insensitive to 4-aminopyridine and barium. In single-channel studies, lemakalim (1 microM) and diazoxide (300 microM) increased the open probability of a 27-pS K+ channel. Openings of these channels decreased with time after patch excision. Application of ADP (1 mM) or ATP (0.1 mM) to the inner surface of the patches reactivated channel openings. The conductance and characteristics of the channels activated by lemakalim were consistent with the properties of KATP. RT-PCR demonstrated the presence of Kir 6.2 and SUR2B transcripts in colonic smooth muscle cells; transcripts for Kir 6.1, SUR1, and SUR2A were not detected. These molecular studies are the first to identify the molecular components of KATP in colonic smooth muscle cells. Together with the electrophysiological experiments, we conclude that KATP channels are expressed in murine colonic smooth muscle cells and suggest that these channels may be involved in dual regulation of resting membrane potential, excitability, and contractility.

Muscarinic regulation of pacemaker frequency in murine gastric interstitial cells of Cajal

Peristaltic contractions in the stomach are regulated by the spread of electrical slow waves from the corpus to the pylorus. Gastric slow waves are generated and propagated by the interstitial cells of Cajal (ICC). All regions distal to the dominant pacemaker area in the corpus are capable of generating slow waves, but orderly gastric peristalsis depends upon a frequency gradient in which the corpus pacemaker frequency exceeds the antral frequency. Cholinergic, muscarinic stimulation enhances pacemaker frequency. We investigated this phenomenon using intact murine gastric muscles and cultured ICC. Acetylcholine (ACh) increased the frequency of slow waves in antrum and corpus muscles. The increase was significantly greater in the antrum. ACh and carbachol (CCh) increased the pacemaker currents in cultured ICC. At high doses of CCh, transient pacemaker currents fused into sustained inward currents that persisted for the duration of stimulation. The effects of CCh were blocked by low doses of the M3 receptor antagonist 1‐dimethyl‐4‐diphenylacetoxypiperidinium. Frequency enhancement by CCh was not affected by forskolin, but the phospholipase C inhibitor U‐73122 inhibited both the increase in frequency and the development of tonic inward currents. 2‐Aminoethyldiphenyl borate also blocked the chronotropic responses to CCh. Inhibitors of protein kinase C did not block responses to CCh. These studies show that mice are an excellent model for studying mechanisms that regulate gastric slow‐wave frequency. CCh, apparently via production of inositol 1,4,5‐trisphosphate, accelerates the frequency of pacemaker activity. High concentrations of CCh may block the entrainment of pacemaker currents, resulting in a tonic inward current.

Anoctamins and gastrointestinal smooth muscle excitability

Interstitial cells of Cajal (ICC) generate electrical pacemaker activity in gastrointestinal smooth muscles. We investigated whether Tmem16a, which encodes anoctamin 1 (ANO1), a Ca2+‐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 (Tmem16atm1Bdh/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 Ca2+, replacement of Ca2+ with Ba2+, or extracellular Ni2+ (30 μm) blocked the inward current. Single Ca2+‐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 (IC50= 4.8 μm). The role of ANO1 in cholinergic responses in ICC was also investigated. Carbachol activated Ca2+‐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.

Important role of mucosal serotonin in colonic propulsion and peristaltic reflexes: in vitro analyses in mice lacking tryptophan hydroxylase 1

•  Previous studies have indicated that neither neuronal nor mucosal 5‐hydroxytryptamine (5‐HT) are important for colonic migrating motor complexes (CMMCs) or faecal pellet propulsion. Therefore, tryptophan hydroxylase 1 knockout (TPH1KO) mice were used to examine the role of mucosal 5‐HT in generating CMMCs and faecal pellet propulsion, as TPH1 is the regulatory enzyme necessary for the synthesis of 5‐HT in enterochromaffin cells in the mucosa. •  Control mice generated a robust CMMC when the mucosa was mechanically stimulated, which was blocked by ondansetron (5‐HT3 antagonist), and could propagate faecal pellets that did not significantly distend the bowel, suggesting that they were propelled by mucosal reflexes in the absence of stretch reflexes. •  TPH1KO mice exhibited no mucosal reflexes, reduced responses to intraluminal distension and propelled only larger faecal pellets, suggesting that they relied upon stretch reflexes alone. •  In control mice, CMMCs, which can propel a faecal pellet, propagated in an oral to anal direction, whereas, in TPH1KO mice, they rarely propagated. •  Both the propagation and amplitude of CMMCs were reduced by ondansetron in control mice, whereas this drug did not affect CMMCs in TPH1KO mice. •  This suggests that 5‐HT release from the mucosa and stretch reflexes are important for normal colonic propulsion.