Generation Of Pacemaker Activity Research Articles

Ca2+ dynamics in interstitial cells: foundational mechanisms for the motor patterns in the gastrointestinal tract.

The gastrointestinal (GI) tract displays multiple motor patterns that move nutrients and wastes through the body. Smooth muscle cells (SMCs) provide the forces necessary for GI motility, but interstitial cells, electrically coupled to SMCs, tune SMC excitability, transduce inputs from enteric motor neurons, and generate pacemaker activity that underlies major motor patterns, such as peristalsis and segmentation. The interstitial cells regulating SMCs are interstitial cells of Cajal (ICC) and PDGF receptor (PDGFR)α+ cells. Together these cells form the SIP syncytium. ICC and PDGFRα+ cells express signature Ca2+-dependent conductances: ICC express Ca2+-activated Cl- channels, encoded by Ano1, that generate inward current, and PDGFRα+ cells express Ca2+-activated K+ channels, encoded by Kcnn3, that generate outward current. The open probabilities of interstitial cell conductances are controlled by Ca2+ release from the endoplasmic reticulum. The resulting Ca2+ transients occur spontaneously in a stochastic manner. Ca2+ transients in ICC induce spontaneous transient inward currents and spontaneous transient depolarizations (STDs). Neurotransmission increases or decreases Ca2+ transients, and the resulting depolarizing or hyperpolarizing responses conduct to other cells in the SIP syncytium. In pacemaker ICC, STDs activate voltage-dependent Ca2+ influx, which initiates a cluster of Ca2+ transients and sustains activation of ANO1 channels and depolarization during slow waves. Regulation of GI motility has traditionally been described as neurogenic and myogenic. Recent advances in understanding Ca2+ handling mechanisms in interstitial cells and how these mechanisms influence motor patterns of the GI tract suggest that the term "myogenic" should be replaced by the term "SIPgenic," as this review discusses.

Propagation of Pacemaker Activity and Peristaltic Contractions in the Mouse Renal Pelvis Rely on Ca2+-activated Cl- Channels and T-Type Ca2+ Channels.

The process of urine removal from the kidney occurs via the renal pelvis (RP). The RP demarcates the beginning of the upper urinary tract and is endowed with smooth muscle cells. Along the RP, organized contraction of smooth muscle cells generates the force required to move urine boluses toward the ureters and bladder. This process is mediated by specialized pacemaker cells that are highly expressed in the proximal RP that generate spontaneous rhythmic electrical activity to drive smooth muscle depolarization. The mechanisms by which peristaltic contractions propagate from the proximal to distal RP are not fully understood. In this study, we utilized a transgenic mouse that expresses the genetically encoded Ca2+ indicator, GCaMP3, under a myosin heavy chain promotor to visualize spreading peristaltic contractions in high spatial detail. Using this approach, we discovered variable effects of L-type Ca2+ channel antagonists on contraction parameters. Inhibition of T-type Ca2+ channels reduced the frequency and propagation distance of contractions. Similarly, antagonizing Ca2+-activated Cl- channels or altering the transmembrane Cl- gradient decreased contractile frequency and significantly inhibited peristaltic propagation. These data suggest that voltage-gated Ca2+ channels are important determinants of contraction initiation and maintain the fidelity of peristalsis as the spreading contraction moves further toward the ureter. Recruitment of Ca2+-activated Cl- channels, likely Anoctamin-1, and T-type Ca2+ channels are required for efficiently conducting the depolarizing current throughout the length of the RP. These mechanisms are necessary for the efficient removal of urine from the kidney.

Autonomous initiation of pacemaking by L-type Cav1.3 calcium channels in mouse sino-atrial myocytes

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Ecole doctorale Background Cardiac pacemaking relies on the spontaneous electrical activity in the right atrium of sino-atrial myocytes (SANCs). Automaticity in SANCs results from a robust interplay of membrane ion channels activity and intracellular calcium dynamics. However, only a fraction of isolated SANCs exhibit rhythmic firing, whereas most SANCs show irregular (dysrhythmic) firing or remain dormant. Purpose To study the capability of L-type Cav1.3 calcium channels to initiate automaticity in dormant SANCs under β-adrenergic stimulation, we used a knock-in mouse strain in which the sensitivity of Cav1.2 α1 subunits to dihydropyridines (DHP) was inactivated (Cav1.2DHP-/-). Methods We performed current and voltage-clamp recordings on isolated SANCs under isoprenaline (ISO, 100 nM) and in the absence or presence of the DHP blocker Nifedipine (Nife, 3 µM). Results Nife significantly reduced the spontaneous firing under ISO perfusion in all rhythmic SANCs (ISO: 447 ± 12, ISO + Nife: 233 ± 25 bpm) and 60% of dysrhythmic SANCs (ISO: 386 ± 12, ISO + Nife: 188 ± 47 bpm) whereas it completely stopped it in the remaining 40% (295 ± 29 bpm to 0). On 25 dormant SANCs, 50% started firing after ISO perfusion (0 to 320 ± 46 bpm). Strikingly, in 75% of them, Nife totally blocked this ISO-induced firing. Interestingly, these cells exhibited a significantly slower rate and a slower slope of the diastolic depolarization under ISO perfusion compared to the remaining 25% dormant SANCs in which Nife only reduced the ISO-induced firing. Moreover, dormant SANCs showed a statistically significant increase in action potential (AP) threshold under ISO compared to dysrhythmic and rhythmic SANCs (dormant: -30.1 ± 2.5, dysrhythmic: -43.3 ± 2.3, rhythmic: -41.2 ± 2.1 mV). No significant difference was observed in the other AP parameters between dormant, dysrhythmic and rhythmic SANCs under ISO. Conclusion Our results seem to point at a difference of expression in ionic channels (Cav1.3, HCN4) within isolated SANCs. Preliminary results on If density support this hypothesis with a lesser density in dormant SANCs compared to dysrhythmic SANCs. These results also tend to indicate that Cav1.3 channels can generate pacemaker activity autonomously, at least in a particular subpopulation of SANCs.

Intrinsic burst-firing in lamina I spinoparabrachial neurons during adolescence

A subset of glutamatergic interneurons in the neonatal spinal superficial dorsal horn (SDH) exhibits intrinsic burst-firing (i.e. ‘pacemaker’ activity), which is tightly regulated by persistent, voltage-gated Na+ channels and classic inward-rectifying K+ (Kir2) channels and downregulated over the course of postnatal development. Ascending lamina I projection neurons targeting the parabrachial nucleus (PB) or periaqueductal gray (PAG) can also display pacemaker activity during early life. However, the degree to which the ionic mechanisms driving pacemaker activity are conserved across different cell types in the spinal dorsal horn, as well as whether the intrinsic bursting is restricted to newborn projection neurons, remains to be elucidated. Using in vitro patch clamp recordings from identified lamina I spinoparabrachial neurons in rat spinal cord slices, here we demonstrate that adolescent projection neurons retain their ability to generate pacemaker activity. In contrast to previous findings in lamina I interneurons, pacemaker projection neurons possessed higher membrane capacitance, lower membrane resistance, and a greater Kir-mediated conductance compared to adjacent spinoparabrachial neurons that lacked intrinsic burst-firing. Nonetheless, as previously seen in interneurons, the bath application of riluzole to block persistent Na+ channels significantly dampened pacemaker activity in projection neurons. Collectively, these results suggest that intrinsic burst-firing in the developing dorsal horn can be generated by multiple combinations of ionic conductances, and highlight the need for further investigation into the mechanisms governing pacemaker activity within the major output neurons of the SDH network.

Ca2+ signaling driving pacemaker activity in submucosal interstitial cells of Cajal in the murine colon

Interstitial cells of Cajal (ICC) generate pacemaker activity responsible for phasic contractions in colonic segmentation and peristalsis. ICC along the submucosal border (ICC-SM) contribute to mixing and more complex patterns of colonic motility. We show the complex patterns of Ca2+ signaling in ICC-SM and the relationship between ICC-SM Ca2+ transients and activation of smooth muscle cells (SMCs) using optogenetic tools. ICC-SM displayed rhythmic firing of Ca2+transients ~ 15 cpm and paced adjacent SMCs. The majority of spontaneous activity occurred in regular Ca2+ transients clusters (CTCs) that propagated through the network. CTCs were organized and dependent upon Ca2+ entry through voltage-dependent Ca2+ conductances, L- and T-type Ca2+ channels. Removal of Ca2+ from the external solution abolished CTCs. Ca2+ release mechanisms reduced the duration and amplitude of Ca2+ transients but did not block CTCs. These data reveal how colonic pacemaker ICC-SM exhibit complex Ca2+-firing patterns and drive smooth muscle activity and overall colonic contractions.

Autonomous initiation of pacemaking by L-type Cav1.3 calcium channels in mouse sino-atrial myocytes

Cardiac pacemaking relies on the spontaneous electrical activity in the right atrium of sino-atrial myocytes (SANCs). Automaticity in SANCs results from a robust interplay of membrane ion channels activity and intracellular calcium dynamics. However, only a fraction of isolated SANCs exhibit rhythmic firing, whereas most SANCs show irregular (dysrhythmic) firing or remain dormant. To study the capability of L-type Cav1.3 calcium channels to initiate automaticity in dormant SANCs under β-adrenergic stimulation we used a knock-in mouse strain in which the sensitivity of Cav1.2 α1 subunits to dihydropyridines (DHP) was inactivated (Cav1.2DHP-/-). We performed current-clamp recordings on isolated SANCs under isoprenaline (ISO, 100 nM) and in the absence or presence of the DHP blocker Nifedipine (Nife, 3 μM). In rhythmic SANCs, Nife significantly reduced the spontaneous firing under ISO perfusion (ISO: 447 ± 12, ISO + Nife: 233 ± 25 bpm). In dysrhythmic SANCs, Nife significantly reduced the spontaneous rate under ISO in 60% of the cells (ISO: 386 ± 12, ISO + Nife: 188 ± 47 bpm) and completely stopped it in 40% (295 ± 29 bpm to 0). On 25 dormant SANCs, 50% started firing after ISO perfusion (0 to 320 ± 46 bpm). Strikingly, in 75% of them, Nife totally blocked this ISO-induced firing. Moreover, dormant SANCs showed a statistically significant increase in action potential (AP) threshold under ISO compared to dysrhythmic and rhythmic SANCs (dormant: −30.1 ± 2.5, dysrhythmic: −43.3 ± 2.3, rhythmic: −41.2 ± 2.1 mV). No significant difference was observed in the other AP parameters between dormant, dysrhythmic and rhythmic SANCs under ISO. Our results indicate that Cav1.3 channels can generate pacemaker activity autonomously, at least in a particular subpopulation of SANCs, while it is responsible of about half of the automaticity in the others.

A novel intramuscular Interstitial Cell of Cajal is a candidate for generating pacemaker activity in the mouse internal anal sphincter

The internal anal sphincter (IAS) generates phasic contractions and tone. Slow waves (SWs) produced by interstitial cells of Cajal (ICC) underlie phasic contractions in other gastrointestinal regions. SWs are also present in the IAS where only intramuscular ICC (ICC-IM) are found, however the evidence linking ICC-IM to SWs is limited. This study examined the possible relationship between ICC-IM and SWs by recording Ca2+ transients in mice expressing a genetically-encoded Ca2+-indicator in ICC (Kit-Cre-GCaMP6f). A role for L-type Ca2+ channels (CavL) and anoctamin 1 (ANO1) was tested since each is essential for SW and tone generation. Two distinct ICC-IM populations were identified. Type I cells (36% of total) displayed localised asynchronous Ca2+ transients not dependent on CavL or ANO1; properties typical of ICC-IM mediating neural responses in other gastrointestinal regions. A second novel sub-type, i.e., Type II cells (64% of total) generated rhythmic, global Ca2+ transients at the SW frequency that were synchronised with neighbouring Type II cells and were abolished following blockade of either CavL or ANO1. Thus, the spatiotemporal characteristics of Type II cells and their dependence upon CavL and ANO1 all suggest that these cells are viable candidates for the generation of SWs and tone in the IAS.

Pacemaker network properties determine intestinal motor pattern behaviour.

Pacemaker network properties determine intestinal motor pattern behaviour.

Conversion of Ca2+ oscillation into propagative electrical signals by Ca2+-activated ion channels and connexin as a reconstituted Ca2+ clock model for the pacemaker activity

Conversion of intracellular Ca2+ signals to electrical activity results in multiple and differing physiological impacts depending on cell types. In some organs such as gastrointestinal and urinary systems, spontaneous Ca2+ oscillation in pacermaker cells can function essentially as a Ca2+ clock mechanism, which has been originally found in pacemaking in sinoatrial node cell of the heart. The conversion of discrete Ca2+ clock events to spontaneous electrical activity is an essential step for the initiation and propagation of pacemaker activity through the multicellular organs resulting in synchronized physiological functions. Here, a model of intracellular signal transduction from a Ca2+ oscillation to initiation of electrical slow waves and their propagation were reconstituted in HEK293 cells. This was accomplished based on ryanodine receptor (RyR) type 3, Ca2+-activated ion channels, i.e. small conductance Ca2+-activated K+ channel (SK2) or Ca2+-activated Cl− channel (TMEM16A), and connexin43 being heterologously co-expressed. The propagation of electrical waves was abolished or substantially reduced by treatment with selective blockers of the expressed channels and 18β-glycyrrhetinic acid, a gap junction inhibitor, respectively. Thus, we demonstrated that the conversion of Ca2+ oscillation to electrical signals with cell to cell propagation can be reconstituted as a model of Ca2+ clock pacemaker activity by combinational expression of critical elements in heterologous expression system.

Characteristics of Ivabradine-Sensitive Currents in Mouse Sinoatrial Node Myocytes

Cardiac pacemaking is driven by spontaneous action potentials (APs) in sinoatrial node myocytes (SAMs). The funny current (If) is thought play a role in the generation of pacemaker activity in SAMs, but the degree to which it contributes is incompletely understood. In this study, we used AP clamp experiments and the If blocker, ivabradine, to investigate the role of If in pacemaking. Spontaneous APs were recorded from acutely-isolated SAMs from mice, and ∼10 s of the AP recording was incorporated into a voltage clamp command. This AP clamp protocol elicited both inward and outward ivabradine-sensitive currents in SAMs. If was defined as the ivabradine-sensitive current remaining in the presence of a pharmacological cocktail of sodium, calcium and potassium channel blockers. Measurable inward ivabradine-sensitive current was evident during diastole in 57% of all cells and was generally larger in cells with the most negative diastolic voltages. Although we found that 30 μM ivabradine also blocked both L- and T-type calcium currents in SAMs, the majority of the ivabradine-sensitive inward current in the AP clamp experiments appeared to be If because control experiments showed that the drug cocktail blocked ∼85% of the calcium currents. Ivabradine also blocked a substantial outward current during the AP upstroke and repolarization phases. This outward ivabradine-sensitive current likely includes a contribution of If, which results from its relatively slow deactivation. However some of the outward ivabradine-sensitive current may also reflect off-target block of potassium currents by ivabradine. We are currently assaying the contribution of potassium currents to the outward ivabradine-sensitive current. Our results suggest that If is active not only during diastole, but also throughout the AP upstroke and repolarization phases of the mouse SAM AP.

AB285. SPR-12 Evidence supporting a pivotal role for intramuscular interstitial cells of Cajal in the generation of pacemaker activity, phasic contractions and tone in the internal anal sphincter

ObjectiveA fundamental property of the internal anal sphincter (IAS) is its ability to generate tone. However, the mechanism underlying tone generation is still controversial. In other GI regions, interstitial cells of Cajal (ICC) have been demonstrated to be the pacemaker cells that generate slow waves (SWs). We have shown that SWs are also present in the IAS along with a population of intramuscular ICC (ICC-IM). However, the identity of the cell that generates SWs in the IAS is still uncertain. We hypothesize that SWs are generated by ICC-IM via a rise in intracellular calcium, activation of calcium-activated chloride channels (ANO1) and voltage-dependent L-type calcium channels (CavL). We further propose that SWs play a critical role in tone development in the IAS through the generation and summation of phasic contractile activity.MethodsCell specific protein expression was examined with immunohistochemistry while gene expression was determined with qPCR on whole muscles and cells isolated with FACS. An inducible Cre/loxP technique was used to express a genetically-encoded Ca2+ biosensor (GCaMP3) in a cell-specific manner. Calcium transients were imaged from the IAS of mice expressing GCaMP3 in ICC (Kit-GCaMP3) and smooth muscle cells (SMC; smMHC-GCaMP3) with an Olympus DSU.ResultsDual labeling immunohistochemistry revealed ANO1 expression in ICC but not SMC while gene expression of ANO1 (Ano1) was 26× greater in FACS-sorted ICC than in sorted SMC. In contrast, gene expression of CavL (Cacna1c) was only 2× greater in SMC than in ICC. Calcium transients were visualized in Kit-GCaMP3+ cells in the circular muscle layer of the IAS. Kit-GCaMP3+ cells exhibited rhythmic whole cell Ca2+ transients that occurred at the same frequency as the SWs we have previously recorded from this muscle. The activity of adjacent Kit-GCaMP3+ cells (<200 µm) was synchronized suggesting coupling between ICC-IM. Rhythmic calcium transients in Kit-GCaMP3+ cells were abolished by removal of extracellular calcium, by the CavL blocker nicardipine (1 µM) and the ANO1 blocker CaCCinh-A01 (3–10 µM). SWs and phasic contractions were also abolished by these blockers. Like SWs, Ca2+ transients in both smMHC-GCaMP3+ and Kit-GCaMP3+ cells were greatest in frequency at the distal extremity of the IAS and declined in the proximal direction. However, when the distal edge of the IAS was removed, both SWs and Ca2+ transients persisted in the remaining muscle although there was a reduction in frequency.ConclusionsThis study provides the first direct evidence that ICC-IM are the pacemaker cells of the IAS. The properties of Ca2+ transients in Kit-GCaMP3+ and smMHC-GCaMP3+ cells are commensurate with the electrical and contractile properties of this muscle. The ability of nicardipine and CaCCinh-A01 to abolish tone, SWs and rhythmic calcium transients in Kit-GCaMP3+ cells suggests a causal relationship between these events. The importance of the distal edge of the IAS in regulating phasic contractions in this muscle is underscored by the decline in both SW and Ca2+ transient frequency and amplitude in the proximal direction indicating that like the heart there is a dominant site of pacemaker activity in the IAS. The observation that the greatest frequency of these events is at the distal edge suggests an important function in ensuring fecal continence.Funding Source(s)NIH DK078736

AB277. SPR-04 Myosalpinx contractions are essential for egg/embryo transport in the oviduct

ObjectiveOviducts or fallopian tubes are smooth muscle lined dynamic tubular organs that connect the ovaries to the uterus and facilitate several physiological processes including gamete transport, fertilization and early embryo development. Several stages of the reproductive process occur exclusively within the oviduct and are subject to physiological regulation. The four main functions of the oviduct include: (I) the transport of the ovum from the ovary to the site of fertilization; (II) the oviduct aids in transport of spermatozoa from the site of deposition to the site of fertilization within the ampulla; (III) the oviduct provides a suitable environment for the egg with secretions along the duct providing protection of the egg from mechanical damage and for maintenance of viability of the egg during its transport; (IV) transportation of the fertilized ovum (embryo) to the uterus where implantation and further development occurs. Given the complexities and the different physiological functions of the oviduct it is not surprising that it is functionally organized into different regions. Dogma in the literature suggests that egg or embryo transport depends mainly on the incessant beat of the ciliated epithelium towards the uterus. However, egg transport may also be dependent upon contractions of the smooth muscle lined wall or myosalpinx. In order to fully understand the physical mechanisms that contribute to the coordinated movement of the egg we investigated the role of the calcium-activated chloride conductance, Ano1 on the generation of pacemaker activity.MethodsVideo imaging and spatio-temporal mapping along with intracellular microelectrode recordings were performed to define the role of myosalpinx in egg/embryo transport. The Cre-recombinase LoxP technology was also utilized to determine the role of the calcium-activated chloride conductance, Ano1 in adult tissues.ResultsEgg transport is highly dependent upon phasic contractions of the smooth muscle lined wall or myosalpinx. The phasic and propulsive contractions of the myosalpinx depend upon specialized ICC, termed ICC-OVI. There is regional dependence on the role of Ano1 in pacemaker activity and that global reduction in Ano1 in mature female mice leads to reduced fertility.ConclusionsThese data further support the role of myosalpinx activity in egg/embryo transport in the oviduct and the importance of Ano1 in the generation of pacemaker activity within the oviduct. A critical role in Ano1 in egg/embryo transport and fertility is also demonstrated.Funding Source(s)NIH RO1 DK57236

Ethanol-Sensitive Pacemaker Neurons in the Mouse External Globus Pallidus

Although ethanol is one of the most widely used drugs, we still lack a full understanding of which neuronal subtypes are affected by this drug. Pacemaker neurons exert powerful control over brain circuit function, but little is known about ethanol effects on these types of neurons. Neurons in the external globus pallidus (GPe) generate pacemaker activity that controls basal ganglia, circuitry associated with habitual and compulsive drug use. We performed patch-clamp recordings from GPe neurons and found that bath application of ethanol dose-dependently decreased the firing rate of low-frequency GPe neurons, but did not alter the firing of high-frequency neurons. GABA or glutamate receptor antagonists did not block the ethanol effect. The GPe is comprised of a heterogeneous population of neurons. We used Lhx6-EGFP and Npas1-tdTm mice strains to identify low-frequency neurons. Lhx6 and Npas1 neurons exhibited decreased firing with ethanol, but only Npas1 neurons were sensitive to 10 mM ethanol. Large-conductance voltage and Ca2+-activated K+ (BK) channel have a key role in the ethanol effect on GPe neurons, as the application of BK channel inhibitors blocked the ethanol-induced firing decrease. Ethanol also increased BK channel open probability measured in single-channel recordings from Npas1-tdTm neurons. In addition, in vivo electrophysiological recordings from GPe showed that ethanol decreased the firing of a large subset of low-frequency neurons. These findings indicate how selectivity of ethanol effects on pacemaker neurons can occur, and enhance our understanding of the mechanisms contributing to acute ethanol effects on the basal ganglia.

Effects of new-generation inhibitors of the calcium-activated chloride channel anoctamin 1 on slow waves in the gastrointestinal tract.

High-throughput screening of compound libraries using genetically encoded fluorescent biosensors has identified several second-generation. low MW inhibitors of the calcium-activated chloride channel anoctamin 1 (CaCC/Ano1). Here we have (i) examined the effects of these Ano1 inhibitors on gastric and intestinal pacemaker activity; (ii) compared the effects of these inhibitors with those of the more classical CaCC inhibitor, 5-nitro-2-(3-phenylpropylalanine) benzoate (NPPB); (ii) examined the mode of action of these compounds on the waveform of pacemaker activity; and (iii) compared differences in the sensitivity between gastric and intestinal pacemaker activity to the Ano1 inhibitors. Using intracellular microelectrode recordings of gastric and intestinal muscle preparations from C57BL/6 mice, the dose-dependent effects of Ano1 inhibitors were examined on spontaneous electrical slow waves. The efficacy of second-generation Ano1 inhibitors on gastric and intestinal pacemaker activity differed significantly. Antral slow waves were more sensitive to these inhibitors than intestinal slow waves. CaCCinh -A01 and benzbromarone were the most potent at inhibiting slow waves in both muscle preparations and more potent than NPPB. Dichlorophene and hexachlorophene were equally potent at inhibiting slow waves. Surprisingly, slow waves were relatively insensitive to T16Ainh -A01 in both preparations. We have identified several second-generation Ano1 inhibitors, blocking gastric and intestinal pacemaker activity. Different sensitivities to Ano1 inhibitors between stomach and intestine suggest the possibility of different splice variants in these two organs or the involvement of other conductances in the generation and propagation of pacemaker activity in these tissues.

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.

The significance of interstitial cells in neurogastroenterology.

Smooth muscle layers of the gastrointestinal tract consist of a heterogeneous population of cells that include enteric neurons, several classes of interstitial cells of mesenchymal origin, a variety of immune cells and smooth muscle cells (SMCs). Over the last number of years the complexity of the interactions between these cell types has begun to emerge. For example, interstitial cells, consisting of both interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor alpha-positive (PDGFRα+) cells generate pacemaker activity throughout the gastrointestinal (GI) tract and also transduce enteric motor nerve signals and mechanosensitivity to adjacent SMCs. ICC and PDGFRα+ cells are electrically coupled to SMCs possibly via gap junctions forming a multicellular functional syncytium termed the SIP syncytium. Cells that make up the SIP syncytium are highly specialized containing unique receptors, ion channels and intracellular signaling pathways that regulate the excitability of GI muscles. The unique role of these cells in coordinating GI motility is evident by the altered motility patterns in animal models where interstitial cell networks are disrupted. Although considerable advances have been made in recent years on our understanding of the roles of these cells within the SIP syncytium, the full physiological functions of these cells and the consequences of their disruption in GI muscles have not been clearly defined. This review gives a synopsis of the history of interstitial cell discovery and highlights recent advances in structural, molecular expression and functional roles of these cells in the GI tract.

Basal cGMP regulates the resting pacemaker potential frequency of cultured mouse colonic interstitial cells of Cajal

Cyclic guanosine 3',5'-monophosphate (cGMP) inhibited the generation of pacemaker activity in interstitial cells of Cajal (ICCs) from the small intestine. However, cGMP role on pacemaker activity in colonic ICCs has not been reported yet. Thus, we investigated the role of cGMP in pacemaker activity regulation by colonic ICCs. We performed a whole-cell patch-clamp and Ca(2+) imaging in cultured ICCs from mouse colon. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) increased the pacemaker potential frequency, whereas zaprinast (an inhibitor of phosphodiesterase) and cell-permeable 8-bromo-cGMP decreased the pacemaker potential frequency. KT-5823 (an inhibitor of protein kinase G [PKG]) did not affect the pacemaker potential. L-N(G)-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide [NO] synthase) increased the pacemaker potential frequency, whereas (±)-S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) decreased the pacemaker potential frequency. Glibenclamide (an ATP-sensitive K(+) channel blocker) did not block the effects of cell-permeable 8-bromo-cGMP and SNAP. Recordings of spontaneous intracellular Ca(2+) ([Ca(2+)]i) oscillations revealed that ODQ and L-NAME increased [Ca(2+)]i oscillations. In contrast, zaprinast, 8-bromo cGMP, and SNAP decreased the [Ca(2+)]i oscillations. Basal cGMP levels regulate the resting pacemaker potential frequency by the alteration on Ca(2+) release via a PKG-independent pathway. Additionally, the endogenous release of NO seems to be responsible maintaining basal cGMP levels in colonic ICCs.

HCN channelopathy and cardiac electrophysiologic dysfunction in genetic and acquired rat epilepsy models

Evidence from animal and human studies indicates that epilepsy can affect cardiac function, although the molecular basis of this remains poorly understood. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate pacemaker activity and modulate cellular excitability in the brain and heart, with altered expression and function associated with epilepsy and cardiomyopathies. Whether HCN expression is altered in the heart in association with epilepsy has not been investigated previously. We studied cardiac electrophysiologic properties and HCN channel subunit expression in rat models of genetic generalized epilepsy (Genetic Absence Epilepsy Rats from Strasbourg, GAERS) and acquired temporal lobe epilepsy (post-status epilepticus SE). We hypothesized that the development of epilepsy is associated with altered cardiac electrophysiologic function and altered cardiac HCN channel expression. Electrocardiography studies were recorded in vivo in rats and in vitro in isolated hearts. Cardiac HCN channel messenger RNA (mRNA) and protein expression were measured using quantitative PCR and Western blotting respectively. Cardiac electrophysiology was significantly altered in adult GAERS, with slower heart rate, shorter QRS duration, longer QTc interval, and greater standard deviation of RR intervals compared to control rats. In the post-SE model, we observed similar interictal changes in several of these parameters, and we also observed consistent and striking bradycardia associated with the onset of ictal activity. Molecular analysis demonstrated significant reductions in cardiac HCN2 mRNA and protein expression in both models, providing a molecular correlate of these electrophysiologic abnormalities. These results demonstrate that ion channelopathies and cardiac dysfunction can develop as a secondary consequence of chronic epilepsy, which may have relevance for the pathophysiology of cardiac dysfunction in patients with epilepsy.

Burst Pacemaker Activity in NCX1 Knockout Mice: Is it Funny Current?

The cardiac Sodium-Calcium exchanger (NCX1) is the dominant Ca efflux protein in SinoAtrial Node (SAN) cells. NCX1 has also been implicated in the generation of pacemaker activity, through the “Ca clock”. To study SAN pacing in the absence of NCX1, we created an atrial-specific NCX1 KO mouse using a Cre/loxP system under the control of the endogenous sarcolipin promoter. NCX1 KO mice have complete AV block and no P waves. The latter is caused by conduction interference between SAN and atria. To test the hypothesis that SAN pacemaking can be maintained by funny current (If) in the absence of NCX1, we recorded Ca transients (using Cal 520 and high speed 2D confocal imaging) in an ex vivo tissuepreparation that included the SAN, right atrium and left atrium. At ∼36°C, the SAN pacemaker rate was 5.3±0.3Hz in WT (n=14) but only 2±0.2Hz (n=17) in KO. The pattern of pacing in KO was characterized by frequent bursts of Ca transients alternating with long pauses. During bursts in KO, the rate was comparable to WT (4.1±0.3Hz). WT SANs (n=5) showed a 70.5±6.1% increase in rate after exposure to the β-adrenergic agonist ISO (10μM), while KO showed no increase in average rate. However, when considering only rate during burst activity in KO, 6 out of 10 KO SANs responded significantly to ISO 10μM (52±16% increase). The specific If inhibitor ivabradine (IVA, 9μM) reduced the spontaneous pacemaker rate in both WT (n=6) and KO (n=8) SANs (44±9% and 58.9±6.8% decrease, respectively), and even during the bursts in the KO (36±17.9% decrease). Thus, NCX1 KO SANs can generate bursts of pacemaker activity similar to WT. These bursts are responsive to both ISO and IVA, consistent with If-mediated pacemaker activity despite the absence of NCX.

Establishment of pacemaker activity in tissues allotransplanted with interstitial cells of Cajal

Loss or disruption of Kit(+) -interstitial cells of Cajal (ICC) capable of generating pacemaker activity has been implicated in the development of numerous gastrointestinal motility disorders. We sought to develop a model where ICC could be allotransplanted into intestines naturally devoid of these cells. Enzymatically dispersed cells from the intestinal tunica muscularis of Kit(+/copGFP) and Kit(V558Δ) /+ gain-of-function mice were allotransplanted into myenteric plexus regions of W/W(V) mutant intestines that lack ICC at the level of the myenteric plexus (ICC-MY) and pacemaker activity. Immunohistochemical analysis fate mapped the development of ICC-MY networks and intracellular microelectrode recordings provided evidence for the development of functional pacemaker activity. Kit(+) -ICC developed into distinct networks at the level of the myenteric plexus in organotypic cultures over 28days and displayed robust rhythmic pacemaker activity. This study demonstrates the feasibility of allotransplantation of ICC into the myenteric region of the small intestine and the establishment of functional pacemaker activity into tissues normally devoid of ICC-MY and slow waves, thus providing a possible basis for the therapeutic treatment of patients where ICC networks have been disrupted due to a variety of pathophysiological conditions.