Cells and ionic conductances contributing to spontaneous activity in bladder and urethral smooth muscle.

Smooth muscle organs of the lower urinary tract comprise the bladder detrusor and urethral wall, which have a reciprocal contractile relationship during urine storage and micturition. As the bladder fills with urine, detrusor smooth muscle cells (DSMCs) remain relaxed to accommodate increases in intravesical pressure while urethral smooth muscle cells (USMCs) sustain tone to occlude the urethral orifice, preventing leakage. While neither organ displays coordinated regular contractions as occurs in small intestine, lymphatics or renal pelvis, they do exhibit patterns of rhythmicity at cellular and tissue levels. In rabbit and guinea-pig urethra, electrical slow waves are recorded from USMCs. This activity is linked to cells expressing vimentin, c-kit and Ca2+-activated Cl- channels, like interstitial cells of Cajal in the gastrointestinal tract. In mouse, USMCs are rhythmically active (firing propagating Ca2+ waves linked to contraction), and this cellular rhythmicity is asynchronous across tissues and summates to form tone. Experiments in mice have failed to demonstrate a voltage-dependent mechanism for regulating this rhythmicity or contractions in vitro, suggesting that urethral tone results from an intrinsic ability of USMCs to 'pace' their own Ca2+ mobilization pathways required for contraction. DSMCs exhibit spontaneous transient contractions, increases in intracellular Ca2+ and action potentials. Consistent across numerous species, including humans, this activity relies on voltage-dependent Ca2+ influx in DSMCs. While interstitial cells are present in the bladder, they do not 'pace' the organ in an excitatory manner. Instead, specialized cells (PDGFRα+ interstitial cells) may 'negatively pace' DSMCs to prevent bladder overexcitability.

Interstitial cell of Cajal-like cells (ICC-LC) exhibit dynamic spontaneous activity but are not functionally innervated in mouse urethra.

Urethral smooth muscle cells (USMC) contract to occlude the internal urethral sphincter during bladder filling. Interstitial cells also exist in urethral smooth muscles and are hypothesized to influence USMC behaviours and neural responses. These cells are similar to Kit+ interstitial cells of Cajal (ICC), which are gastrointestinal pacemakers and neuroeffectors. Isolated urethral ICC-like cells (ICC-LC) exhibit spontaneous intracellular Ca2+ signalling behaviours that suggest these cells may serve as pacemakers or neuromodulators similar to ICC in the gut, although observation and direct stimulation of ICC-LC within intact urethral tissues is lacking. We used mice with cell-specific expression of the Ca2+ indicator, GCaMP6f, driven off the endogenous promoter for Kit (Kit-GCaMP6f mice) to identify ICC-LC in situ within urethra muscles and to characterize spontaneous and nerve-evoked Ca2+ signalling. ICC-LC generated Ca2+ waves spontaneously that propagated on average 40.1 ± 0.7 µm, with varying amplitudes, durations, and spatial spread. These events originated from multiple firing sites in cells and the activity between sites was not coordinated. ICC-LC in urethra formed clusters but not interconnected networks. No evidence for entrainment of Ca2+ signalling between ICC-LC was obtained. Ca2+ events in ICC-LC were unaffected by nifedipine but were abolished by cyclopiazonic acid and decreased by an antagonist of Orai Ca2+ channels (GSK-7975A). Phenylephrine increased Ca2+ event frequency but a nitric oxide donor (DEA-NONOate) had no effect. Electrical field stimulation (EFS, 10 Hz) of intrinsic nerves, which evoked contractions of urethral rings and increased Ca2+ event firing in USMC, failed to evoke responses in ICC-LC. Our data suggest that urethral ICC-LC are spontaneously active but are not regulated by autonomic neurons.

Diversifying laboratory assessment modes broadens engagement with practical competencies in life science students.

Laboratory practicals in life science subjects are traditionally assessed by written reports that reflect disciplinary norms for documenting experimental activities. However, the exclusive application of this assessment has the potential to engage only a narrow range of competencies. In this study, we explored how multiple modes of laboratory assessment might affect student perceptions of learned skills in a life science module. We hypothesized that while a mixture of assessments may not impact student summative performance, it might positively influence student perceptions of different skills that varied assessments allowed them to practice. This was informed by universal design for learning and teaching for understanding frameworks. In our study, in a third-year Bioscience program, written reports were complemented with group presentations and online quizzes via Moodle. Anonymous surveys evaluated whether this expanded portfolio of assessments promoted awareness of, and engagement with, a broader range of practical competencies. Aspects that influenced student preferences in assessment mode included time limitations, time investment, ability to practice new skills, links with lecture material, and experience of assessment anxiety. In particular, presentations were highlighted as promoting collaboration and communication and the quiz as an effective means of diversifying assessment schedules. A key takeaway from students was that while reports were important, an overreliance on them was detrimental. This study suggests that undergraduate life science students can benefit significantly from a holistic assessment strategy that complements reports with performance-based approaches that incorporate broader competencies and allow for greater student engagement and expression in undergraduate modules.NEW & NOTEWORTHY This study suggests that undergraduate life science students can benefit significantly from a holistic assessment strategy that complements reports with performance-based approaches that incorporate broader competencies and allow for greater student engagement and expression in undergraduate modules.

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.

The TMEM16A blockers benzbromarone and MONNA cause intracellular Ca2+-release in mouse bronchial smooth muscle cells.

We investigated effects of TMEM16A blockers benzbromarone, MONNA, CaCCinhA01 and Ani9 on isometric contractions in mouse bronchial rings and on intracellular calcium in isolated bronchial myocytes. Separate concentrations of carbachol (0.1-10 µM) were applied for 10 min periods to bronchial rings, producing concentration-dependent contractions that were well maintained throughout each application period. Benzbromarone (1 µM) markedly reduced the contractions with a more pronounced effect on their sustained component (at 10 min) compared to their initial component (at 2 min). Iberiotoxin (0.3 µM) enhanced the contractions, but they were still blocked by benzbromarone. MONNA (3 µM) and CaCCinhA01 (10 µM) had similar effects to benzbromarone, but were less potent. In contrast, Ani9 (10 µM) had no effect on carbachol-induced contractions. Confocal imaging revealed that benzbromarone (0.3 µM), MONNA (1 µM) and CaCCinhA01 (10 µM) increased intracellular calcium in isolated myocytes loaded with Fluo-4AM. In contrast, Ani9 (10 µM) had no effect on intracellular calcium. Benzbromarone and MONNA also increased calcium in calcium-free extracellular solution, but failed to do so when intracellular stores were discharged with caffeine (10 mM). Caffeine was unable to cause further discharge of the store when applied in the presence of benzbromarone. Ryanodine (100 µM) blocked the ability of benzbromarone (0.3 µM) to increase calcium, while tetracaine (100 µM) reversibly reduced the rise in calcium induced by benzbromarone. We conclude that benzbromarone and MONNA caused intracellular calcium release, probably by opening ryanodine receptors. Their ability to block carbachol contractions was likely due to this off-target effect.

Ca2+ Signaling Is the Basis for Pacemaker Activity and Neurotransduction in Interstitial Cells of the GI Tract.

Years ago gastrointestinal motility was thought to be due to interactions between enteric nerves and smooth muscle cells (SMCs) in the tunica muscularis. Thus, regulatory mechanisms controlling motility were either myogenic or neurogenic. Now we know that populations of interstitial cells, c-Kit+ (interstitial cells of Cajal or ICC), and PDGFRα+ cells (formerly "fibroblast-like" cells) are electrically coupled to SMCs, forming the SIP syncytium. Pacemaker and neurotransduction functions are provided by interstitial cells through Ca2+ release from the endoplasmic reticulum (ER) and activation of Ca2+-activated ion channels in the plasma membrane (PM). ICC express Ca2+-activated Cl- channels encoded by Ano1. When activated, Ano1 channels produce inward current and, therefore, depolarizing or excitatory effects in the SIP syncytium. PDGFRα+ cells express Ca2+-activated K+ channels encoded by Kcnn3. These channels generate outward current when activated and hyperpolarizing or membrane-stabilizing effects in the SIP syncytium. Inputs from enteric and sympathetic neurons regulate Ca2+ transients in ICC and PDGFRα+ cells, and currents activated in these cells conduct to SMCs and regulate contractile behaviors. ICC also serve as pacemakers, generating slow waves that are the electrophysiological basis for gastric peristalsis and intestinal segmentation. Pacemaker types of ICC express voltage-dependent Ca2+ conductances that organize Ca2+ transients, and therefore Ano1 channel openings, into clusters that define the amplitude and duration of slow waves. Ca2+ handling mechanisms are at the heart of interstitial cell function, yet little is known about what happens to Ca2+ dynamics in these cells in GI motility disorders.

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.

Ca2+ -activated Cl- channels (TMEM16A) underlie spontaneous electrical activity in isolated mouse corpus cavernosum smooth muscle cells.

Penile detumescence is maintained by tonic contraction of corpus cavernosum smooth muscle cells (CCSMC), but the underlying mechanisms have not been fully elucidated. The purpose of this study was to characterize the mechanisms underlying activation of TMEM16A Ca2+ -activated Cl- channels in freshly isolated murine CCSMC. Male C57BL/6 mice aged 10-18 weeks were euthanized via intraperitoneal injection of sodium pentobarbital (100 mg.kg-1 ). Whole-cell patch clamp, pharmacological, and immunocytochemical experiments were performed on isolated CCSM. Tension measurements were performed in whole tissue. TMEM16A expression in murine corpus cavernosum was confirmed using immunocytochemistry. Isolated CCSMC developed spontaneous transient inward currents (STICs) under voltage clamp and spontaneous transient depolarizations (STDs) in current clamp mode of the whole cell, perforated patch clamp technique. STICs reversed close to the predicted Cl- equilibrium potential and both STICs and STDs were blocked by the TMEM16A channel blockers, Ani9 and CaCC(inh)-A01. These events were also blocked by GSK7975A (ORAI inhibitor), cyclopiazonic acid (CPA, sarcoplasmic reticulum [SR] Ca2+- ATPase blocker), tetracaine (RyR blocker), and 2APB (IP3 R blocker), suggesting that they were dependent on Ca2+ release from intracellular Ca2+ stores. Nifedipine (L-type Ca2+ channel blocker) did not affect STICs, but reduced the duration of STDs. Phenylephrine induced transient depolarizations and transient inward currents which were blocked by Ani9. Similarly, phenylephrine induced phasic contractions of intact corpus cavernosum muscle strips and these events were also inhibited by Ani9. This study suggests that contraction of CCSM is regulated by activation of TMEM16A channels and therefore inhibition of these channels could lead to penile erection.

Calcium transients in intramuscular interstitial cells of Cajal of the murine gastric fundus and their regulation by neuroeffector transmission.

Enteric neurotransmission is critical for coordinating motility throughout the gastrointestinal (GI) tract. However, there is considerable controversy regarding the cells that are responsible for the transduction of these neural inputs. In the present study, utilization of a cell-specific calcium biosensor GCaMP6f, the spontaneous activity and neuroeffector responses of intramuscular ICC (ICC-IM) to motor neural inputs was examined. Simultaneous intracellular microelectrode recordings and high-speed video-imaging during nerve stimulation was used to reveal the temporal relationship between changes in intracellular Ca2+ and post-junctional electrical responses to neural stimulation. ICC-IM were highly active, generating intracellular Ca2+ -transients that occurred stochastically, from multiple independent sites in single ICC-IM. Ca2+ -transients were not entrained in single ICC-IM or between neighbouring ICC-IM. Activation of enteric motor neurons produced a dominant inhibitory response that abolished Ca2+ -transients in ICC-IM. This inhibitory response was often preceded by a summation of Ca2+ -transients that led to a global rise in Ca2+ . Individual ICC-IM responded to nerve stimulation by a global rise in Ca2+ followed by inhibition of Ca2+ -transients. The inhibition of Ca2+ -transients was blocked by the nitric oxide synthase antagonist l-NNA. The global rise in intracellular Ca2+ was inhibited by the muscarinic antagonist, atropine. Simultaneous intracellular microelectrode recordings with video-imaging revealed that the rise in Ca2+ was temporally associated with rapid excitatory junction potentials and the inhibition of Ca2+ -transients with inhibitory junction potentials. These data support the premise of serial innervation of ICC-IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach. KEY POINTS: The cells responsible for mediating enteric neuroeffector transmission remain controversial. In the stomach intramuscular interstitial cells of Cajal (ICC-IM) were the first ICC reported to receive cholinergic and nitrergic neural inputs. Utilization of a cell specific calcium biosensor, GCaMP6f, the activity, and neuroeffector responses of ICC-IM were examined. ICC-IM were highly active, generating stochastic intracellular Ca2+ -transients. Stimulation of enteric motor nerves abolished Ca2+ -transients in ICC-IM. This inhibitory response was preceded by a global rise in intracellular Ca2+ . Individual ICC-IM responded to nerve stimulation with a rise in Ca2+ followed by inhibition of Ca2+ -transients. Inhibition of Ca2+ -transients was blocked by the nitric oxide synthase antagonist l-NNA. The global rise in Ca2+ was inhibited by the muscarinic antagonist atropine. Simultaneous intracellular recordings with video imaging revealed that the global rise in intracellular Ca2+ and inhibition of Ca2+ -transients was temporally associated with rapid excitatory junction potentials followed by more sustained inhibitory junction potentials. The data presented support the premise of serial innervation of ICC-IM in excitatory and inhibitory neuroeffector transmission in the proximal stomach.

Insights on gastrointestinal motility through the use of optogenetic sensors and actuators.

The muscularis of the gastrointestinal (GI) tract consists of smooth muscle cells (SMCs) and various populations of interstitial cells of Cajal (ICC), platelet-derived growth factor receptor α+ (PDGFRα+ ) cells, as well as excitatory and inhibitory enteric motor nerves. SMCs, ICC and PDGFRα+ cells form an electrically coupled syncytium, which together with inputs from the enteric nervous system (ENS) regulates GI motility. Early studies evaluating Ca2+ signalling behaviours in the GI tract relied upon indiscriminate loading of tissues with Ca2+ dyes. These methods lacked the means to study activity in specific cells of interest without encountering contamination from other cells within the preparation. Development of mice expressing optogenetic sensors (green calmodulin fusion protein (GCaMP), red calmodulin fusion protein (RCaMP)) has allowed visualization of Ca2+ signalling behaviours in a cell specific manner. Additionally, availability of mice expressing optogenetic modulators (channelrhodopsins or halorhodospins) has allowed manipulation of specific signalling pathways using light. GCaMP-expressing animals have been used to characterize Ca2+ signalling behaviours of distinct classes of ICC and SMCs throughout the GI musculature. These findings illustrate how Ca2+ signalling in ICC is fundamental in GI muscles, contributing to tone in sphincters, pacemaker activity in rhythmic muscles and relaying enteric signals to SMCs. Animals that express channelrhodopsin in specific neuronal populations have been used to map neural circuitry and to examine post junctional neural effects on GI motility. Thus, optogenetic approaches provide a novel means to examine the contribution of specific cell types to the regulation of motility patterns within complex multi-cellular systems.

Propulsive colonic contractions are mediated by inhibition-driven poststimulus responses that originate in interstitial cells of Cajal.

The peristaltic reflex is a fundamental behavior of the gastrointestinal (GI) tract in which mucosal stimulation activates propulsive contractions. The reflex occurs by stimulation of intrinsic primary afferent neurons with cell bodies in the myenteric plexus and projections to the lamina propria, distribution of information by interneurons, and activation of muscle motor neurons. The current concept is that excitatory cholinergic motor neurons are activated proximal to and inhibitory neurons are activated distal to the stimulus site. We found that atropine reduced, but did not block, colonic migrating motor complexes (CMMCs) in mouse, monkey, and human colons, suggesting a mechanism other than one activated by cholinergic neurons is involved in the generation/propagation of CMMCs. CMMCs were activated after a period of nerve stimulation in colons of each species, suggesting that the propulsive contractions of CMMCs may be due to the poststimulus excitation that follows inhibitory neural responses. Blocking nitrergic neurotransmission inhibited poststimulus excitation in muscle strips and blocked CMMCs in intact colons. Our data demonstrate that poststimulus excitation is due to increased Ca2+ transients in colonic interstitial cells of Cajal (ICC) following cessation of nitrergic, cyclic guanosine monophosphate (cGMP)-dependent inhibitory responses. The increase in Ca2+ transients after nitrergic responses activates a Ca2+-activated Cl− conductance, encoded by Ano1, in ICC. Antagonists of ANO1 channels inhibit poststimulus depolarizations in colonic muscles and CMMCs in intact colons. The poststimulus excitatory responses in ICC are linked to cGMP-inhibited cyclic adenosine monophosphate (cAMP) phosphodiesterase 3a and cAMP-dependent effects. These data suggest alternative mechanisms for generation and propagation of CMMCs in the colon.

Ca2+ signalling in interstitial cells of Cajal contributes to generation and maintenance of tone in mouse and monkey lower oesophageal sphincters.

The lower oesophageal sphincter (LES) generates tone and prevents reflux of gastric contents. LES smooth muscle cells (SMCs) are relatively depolarised, facilitating activation of Cav 1.2 channels to sustain contractile tone. We hypothesised that intramuscular interstitial cells of Cajal (ICC-IM), through activation of Ca2+ -activated Cl- channels (ANO1), set membrane potentials of SMCs favourable for activation of Cav 1.2 channels. In some gastrointestinal muscles, ANO1 channels in ICC-IM are activated by Ca2+ transients, but no studies have examined Ca2+ dynamics in ICC-IM within the LES. Immunohistochemistry and qPCR were used to determine expression of key proteins and genes in ICC-IM and SMCs. These studies revealed that Ano1 and its gene product, ANO1, are expressed in c-Kit+ cells (ICC-IM) in mouse and monkey LES clasp muscles. Ca2+ signalling was imaged in situ, using mice expressing GCaMP6f specifically in ICC (Kit-KI-GCaMP6f). ICC-IM exhibited spontaneous Ca2+ transients from multiple firing sites. Ca2+ transients were abolished by cyclopiazonic acid or caffeine but were unaffected by tetracaine or nifedipine. Maintenance of Ca2+ transients depended on Ca2+ influx and store reloading, as Ca2+ transient frequency was reduced in Ca2+ free solution or by Orai antagonist. Spontaneous tone of LES muscles from mouse and monkey was reduced ∼80% either by Ani9, an ANO1 antagonist or by the Cav 1.2 channel antagonist nifedipine. Membrane hyperpolarisation occurred in the presence of Ani9. These data suggest that intracellular Ca2+ activates ANO1 channels in ICC-IM in the LES. Coupling of ICC-IM to SMCs drives depolarisation, activation of Cav 1.2 channels, Ca2+ entry and contractile tone. KEY POINTS: The lower oesophageal sphincter (LES) generates contractile tone preventing reflux of gastric contents into the oesophagus. LES smooth muscle cells (SMCs) display depolarised membrane potentials facilitating activation of L-type Ca2+ channels. Interstitial cells of Cajal (ICC) express Ca2+ -activated Cl- channels encoded by Ano1 in mouse and monkey LES. Ca2+ signalling in ICC activates ANO1 currents in ICC. ICC displayed spontaneous Ca2+ transients in mice from multiple firing sites in each cell and no entrainment of Ca2+ firing between sites or between cells. Inhibition of ANO1 channels with a specific antagonist caused hyperpolarisation of mouse LES and inhibition of tone in monkey and mouse LES muscles. Our data suggest a novel mechanism for LES tone in which Ca2+ transient activation of ANO1 channels in ICC generates depolarising inward currents that conduct to SMCs to activate L-type Ca2+ currents, Ca2+ entry and contractile tone.

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning.

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

Role of Ano1 Ca2+-activated Cl- channels in generating urethral tone.

Urinary continence is maintained in the lower urinary tract by the contracture of urethral sphincters, including smooth muscle of the internal urethral sphincter. These contractions occlude the urethral lumen, preventing urine leakage from the bladder to the exterior. Over the past 20 years, research on the ionic conductances that contribute to urethral smooth muscle contractility has greatly accelerated. A debate has emerged over the role of interstitial cell of Cajal (ICC)-like cells in the urethra and their expression of Ca2+-activated Cl- channels encoded by anoctamin-1 [Ano1; transmembrane member 16 A (Tmem16a) gene]. It has been proposed that Ano1 channels expressed in urethral ICC serve as a source of depolarization for smooth muscle cells, increasing their excitability and contributing to tone. Although a clear role for Ano1 channels expressed in ICC is evident in other smooth muscle organs, such as the gastrointestinal tract, the role of these channels in the urethra is unclear, owing to differences in the species (rabbit, rat, guinea pig, sheep, and mouse) examined and experimental approaches by different groups. The importance of clarifying this situation is evident as effective targeting of Ano1 channels may lead to new treatments for urinary incontinence. In this review, we summarize the key findings from different species on the role of ICC and Ano1 channels in urethral contractility. Finally, we outline proposals for clarifying this controversial and important topic by addressing how cell-specific optogenetic and inducible cell-specific genetic deletion strategies coupled with advances in Ano1 channel pharmacology may clarify this area in future studies.NEW & NOTEWORTHY Studies from the rabbit have shown that anoctamin-1 (Ano1) channels expressed in urethral interstitial cells of Cajal (ICC) serve as a source of depolarization for smooth muscle cells, increasing excitability and tone. However, the role of urethral Ano1 channels is unclear, owing to differences in the species examined and experimental approaches. We summarize findings from different species on the role of urethral ICC and Ano1 channels in urethral contractility and outline proposals for clarifying this topic using cell-specific optogenetic approaches.

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.

The intracellular Ca2+ release channel TRPML1 regulates lower urinary tract smooth muscle contractility.

TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel that is predominantly localized to the membranes of late endosomes and lysosomes (LELs). Intracellular release of Ca2+ through TRPML1 is thought to be pivotal for maintenance of intravesicular acidic pH as well as the maturation, fusion, and trafficking of LELs. Interestingly, genetic ablation of TRPML1 in mice (Mcoln1-/- ) induces a hyperdistended/hypertrophic bladder phenotype. Here, we investigated this phenomenon further by exploring an unconventional role for TRPML1 channels in the regulation of Ca2+-signaling activity and contractility in bladder and urethral smooth muscle cells (SMCs). Four-dimensional (4D) lattice light-sheet live-cell imaging showed that the majority of LELs in freshly isolated bladder SMCs were essentially immobile. Superresolution microscopy revealed distinct nanoscale colocalization of LEL-expressing TRPML1 channels with ryanodine type 2 receptors (RyR2) in bladder SMCs. Spontaneous intracellular release of Ca2+ from the sarcoplasmic reticulum (SR) through RyR2 generates localized elevations of Ca2+ ("Ca2+ sparks") that activate plasmalemmal large-conductance Ca2+-activated K+ (BK) channels, a critical negative feedback mechanism that regulates smooth muscle contractility. This mechanism was impaired in Mcoln1-/- mice, which showed diminished spontaneous Ca2+ sparks and BK channel activity in bladder and urethra SMCs. Additionally, ex vivo contractility experiments showed that loss of Ca2+ spark-BK channel signaling in Mcoln1-/- mice rendered both bladder and urethra smooth muscle hypercontractile. Voiding activity analyses revealed bladder overactivity in Mcoln1-/- mice. We conclude that TRPML1 is critically important for Ca2+ spark signaling, and thus regulation of contractility and function, in lower urinary tract SMCs.

A high throughput machine-learning driven analysis of Ca2+ spatio-temporal maps.

High-resolution Ca2+ imaging to study cellular Ca2+ behaviors has led to the creation of large datasets with a profound need for standardized and accurate analysis. To analyze these datasets, spatio-temporal maps (STMaps) that allow for 2D visualization of Ca2+ signals as a function of time and space are often used. Methods of STMap analysis rely on a highly arduous process of user defined segmentation and event-based data retrieval. These methods are often time consuming, lack accuracy, and are extremely variable between users. We designed a novel automated machine-learning based plugin for the analysis of Ca2+ STMaps (STMapAuto). The plugin includes optimized tools for Ca2+ signal preprocessing, automated segmentation, and automated extraction of key Ca2+ event information such as duration, spatial spread, frequency, propagation angle, and intensity in a variety of cell types including the Interstitial cells of Cajal (ICC). The plugin is fully implemented in Fiji and able to accurately detect and expeditiously quantify Ca2+ transient parameters from ICC. The plugin's speed of analysis of large-datasets was 197-fold faster than the commonly used single pixel-line method of analysis. The automated machine-learning based plugin described dramatically reduces opportunities for user error and provides a consistent method to allow high-throughput analysis of STMap datasets.

TRPML1 channels initiate Ca2+ sparks in vascular smooth muscle cells.

TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel localized to the membranes of endosomes and lysosomes and is not present or functional on the plasma membrane. Ca2+ released from endosomes and lysosomes into the cytosol through TRPML1 channels is vital for trafficking, acidification, and other basic functions of these organelles. Here, we investigated the function of TRPML1 channels in fully differentiated contractile vascular smooth muscle cells (SMCs). In live-cell confocal imaging studies, we found that most endosomes and lysosomes in freshly isolated SMCs from cerebral arteries were essentially immobile. Using nanoscale super-resolution microscopy, we found that TRPML1 channels present in late endosomes and lysosomes formed stable complexes with type 2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Spontaneous Ca2+ signals resulting from the release of SR Ca2+ through RyR2s ("Ca2+ sparks") and corresponding Ca2+-activated K+ channel activity are critically important for balancing vasoconstriction. We found that these signals were essentially absent in SMCs from TRPML1-knockout (Mcoln1-/- ) mice. Using ex vivo pressure myography, we found that loss of this critical signaling cascade exaggerated the vasoconstrictor responses of cerebral and mesenteric resistance arteries. In vivo radiotelemetry studies showed that Mcoln1-/- mice were spontaneously hypertensive. We conclude that TRPML1 is crucial for the initiation of Ca2+ sparks in SMCs and the regulation of vascular contractility and blood pressure.

Excitatory cholinergic responses in mouse colon intramuscular interstitial cells of Cajal are due to enhanced Ca2+ release via M3 receptor activation.

Colonic intramuscular interstitial cells of Cajal (ICC-IM) are associated with cholinergic varicosities, suggesting a role in mediating excitatory neurotransmission. Ca2+ release in ICC-IM activates Ano1, a Ca2+ -activated Cl- conductance, causing tissue depolarization and increased smooth muscle excitability. We employed Ca2+ imaging of colonic ICC-IM in situ, using mice expressing GCaMP6f in ICC to evaluate ICC-IM responses to excitatory neurotransmission. Expression of muscarinic type 2, 3 (M2 , M3 ), and NK1 receptors were enriched in ICC-IM. NK1 receptor agonists had minimal effects on ICC-IM, whereas neostigmine and carbachol increased Ca2+ transients. These effects were reversed by DAU 5884 (M3 receptor antagonist) but not AF-DX 116 (M2 receptor antagonist). Electrical field stimulation (EFS) in the presence of L-NNA and MRS 2500 enhanced ICC-IM Ca2+ transients. Responses were blocked by atropine or DAU 5884, but not AF-DX 116. ICC-IM responses to EFS were ablated by inhibiting Ca2+ stores with cyclopiazonic acid and reduced by inhibiting Ca2+ influx via Orai channels. Contractions induced by EFS were reduced by an Ano1 channel antagonist, abolished by DAU 5884, and unaffected by AF-DX 116. Colonic ICC-IM receive excitatory inputs from cholinergic neurons via M3 receptor activation. Enhancing ICC-IM Ca2+ release and Ano1 activation contributes to excitatory responses of colonic muscles.