The protein is formed by a barrel of 19 beta-sheets with an N-terminal helix lining the inner pore. Despite its large diameter, the channel can change its selectivity for ions and metabolites based on its open state to regulate transport into and out of mitochondria. VDAC was shown to be regulated by a variety of cellular factors and molecular partners including proteins, lipids and ions. Although the physiological importance of many of these modulatory effects are well described, the binding sites for molecular partners are still largely unknown. The highly symmetrical and sleek structure of the channel makes predictions of functional moieties difficult. However, one residue repeatedly sticks out when reviewing VDAC literature. A glutamate at position 73 (E73) located on the outside of the channel facing the hydrophobic membrane environment was repeatedly proposed to be involved in channel regulation on multiple levels. Here, we review the distinct hypothesized roles of E73 and summarize the open questions around this mysterious residue.The voltage-dependent anion channel (VDAC) is the main passageway for ions and metabolites over the outer mitochondrial membrane. It was associated with many physiological processes, including apoptosis and modulation of intracellular Ca It is the main passageway over the outer mitochondrial membrane and conducts ions and small metabolites into and out of mitochondria. Higher organisms express 3 isoforms, VDAC1-3, whose distinct roles are still under debate. While VDAC1, as the best researched isoform, was described to be a major regulator of apoptosis and a regulator of cellular energetics by conducting ATP, the contribution of VDAC2 and 3 to these processes are less clear. Both VDAC1 and VDAC2 were described to be regulators of mitochondrial CaAlthough high resolution structures are so far only available for VDAC1  and VDACWhen inserted into planar lipid bilayers, VDAC resides in an anion-selective, 4nS high-conductance state at potentials around 0 mV, shows vigorous gating behavior at test potentials around ±20–40 mV and resides in several more cation selective low-conductance states (around 2 nS) at potential larger then 50 mV or lower then −50 mV ,10,11. A2+ concentration [2+ signaling.Gating of the channel was shown to be influenced by various factors, such as the lipidic environment ,13,14, Pntration  or the pntration ,18 or mentration . PhysiolDespite several reports about regulation of the channel through the aforementioned factors, the molecular underpinnings still remain largely elusive, especially because the highly symmetrical and very sleek structure of VDAC makes identification of functionally relevant moieties in the channel difficult. Most reports focus on the N-terminal domain, including the helix as a relevant structure for channel gating and interaction with other proteins. As such, a lysine-rich motif in the helix was suggested to guide ATP permeation , while a2+ binding and permeation [On the other hand, motifs in the barrel itself are not well characterized, with the exception of one prominent amino acid, a glutamate at position 73 (E73). This glutamate was first described in 1993 as an important residue for the binding of hexokinase . In furtrmeation , lipid brmeation ,25, and rmeation . HoweverLooking at the high-resolution structure of VDAC, E73 is located in the fourth β-sheet of the transmembrane barrel with its side chain located on the outside of the barrel and sticking into the middle plane of the hydrophobic membrane environment . The areThis flexibility was proposed to be, at least in part, also induced by E73: molecular dynamics simulations revealed that the charge of E73 enhances protein dynamics of the area comprising the first four to six β-sheets of the barrel (with E73 being located in the fourth β-sheet) . RenderiHowever, in later experiments performed in planar lipid bilayers, the exchange of E73 for glutamine or alanine revealed no changes in the voltage-induced gating of recombinant VDAC1 , contradOne potential binding partner could be the channel itself; several lines of evidence indicate that dimerization of VDAC serves as a mode of channel regulation in apoptosis and metabolic control ,35. BergTaken together, it appears unlikely that E73 can influence channel gating in its apo-state , but dim2+. Mitochondria can take up vast amounts of Ca2+ and the uptake and release of Ca2+ from mitochondria is a highly regulated process. Disturbances in mitochondrial Ca2+ uptake are associated with human diseases such as cardiac, metabolic and neurological diseases [2+ uptake was proposed to be a promising pharmacological target structure [One of the most researched interaction partners of VDAC is Cadiseases ,37, as wdiseases ,39, and tructure ,41.2+, VDAC is the main passageway for Ca2+ over the OMM, and several reports suggest a regulation of mitochondrial Ca2+ uptake at this stage [2+ transfer from cellular organelles into mitochondria. A tight contact between lysosomes and mitochondria exists and disruption of this cross-talk was shown to be associated with lysosomal storage diseases. The direct coupling is essential for the transfer of Ca2+ from lysosomes into mitochondria via a TRPML1-VDAC1 axis [While in the inner mitochondrial membrane, the mitochondrial calcium uniporter complex (MCUC) is the main uptake route for Cais stage ,42. Spec2+ store. This network involves a very close interaction between the SR Ca2+ release channel, the ryanodine receptor (RyR) and VDAC2 [2+ uptake near the SR Ca2+ release sites. This mechanism was shown to modulate intracellular Ca2+ signals during excitation-contraction coupling, and to buffer erratic Ca2+ release during diastole to prevent arrhythmogenesis [2+ transfer between the SR and mitochondria in cardiomyocytes and as such to be protective against arrhythmia [2+ transfer in cardiomyocytes, and the overexpression of wild-type VDAC2, but not E73Q, restored rhythmic cardiac contractions in a zebrafish arrhythmia model.Similarly, in cardiomyocytes, mitochondria form close contact sites with the sarcoplasmic reticulum, the main cellular Cand VDAC2  and faciogenesis ,45. Interhythmia . The exp2+ flux through VDAC: on the one hand, VDAC was suggested to change its permeability for Ca2+ depending on its open state [2+ binding to the channel at concentrations larger then 600 nM was itself described to be a modulator of channel gating and thus Ca2+ permeation [Two mechanisms are conceivable to be relevant for a regulation of Caen state ,48, whilrmeation .2+ currents [2+-mediated modulation of the channel or a role in Ca2+-mediated interaction of the channel with regulatory partners appears most likely.As outlined above, a direct role of E73 in channel gating appears unlikely, as lipid bilayer experiments demonstrated that E73 is not involved in the regulation of channel gating, at least not for monomers of the apo-channel . Furthercurrents ,48. Howecurrents   [2+ competes with 25 µM azido ruthenium (AzRu) for a binding site involving E73 and E202 in a dose-dependent manner with a half-maximal effect at 100 µM [2+ inside this hydrophobic environment. Ujwal et al. concluded that VDAC could only hold Ca2+ when two monomers of the channel assemble in an antiparallel dimer and only under very high concentrations of Ca2+, both of which are not expected to appear under physiological conditions [For both, a binding site for Caed (RuR) , the samt 100 µM . The mutt 100 µM , E73 fac2+ uptake, which is directly relevant for human diseases and the treatment thereof; however, the molecular underpinnings of the VDAC-Ca2+ interaction remain elusive. With the notion that E73 was described not to influence gating behavior or ion selectivity and the absence of a defined Ca2+ binding site involving E73, it remains unclear how E73 can control Ca2+ flux through VDAC at a molecular level  can compete out the VDAC-hexokinase interaction , De Pintand DCCP . They fope VDAC1  (Table 1pe VDAC1 . ExperimComing back to the role of cholesterol on VDAC function, it is of note that an interplay of VDAC, cholesterol and hexokinase at E73 was described; as such, the membrane cholesterol content was shown to be enriched in carcinoma cells, which was associated with a reduction in oxidative phosphorylation. A protein complex involving VDAC was shown to be accountable for this process. The overexpression of VDAC E73Q in contrast increased oxidative phosphorylation and reduces membrane cholesterol content .2+ might be involved in this process.Taken together, the interaction of VDAC with hexokinase is essential for the regulation of the cellular metabolism, in particular the change from oxidative phosphorylation to enhanced glycolysis in cancer cells. This interaction is mediated by E73 and is maybe the most intensively researched interaction of VDAC with a molecular partner. A reduction in channel conductance together with downstream effects, such as a reduction of cholesterol content and protection against apoptosis, are associated with this interaction. However, most of these experiments were conducted using overexpression systems, and factors regulating the amount of hexokinase being bound to VDAC under physiological conditions are still unknown. The lipidic environment, intracellular pH, and cellular ions such as CaTaken together, multiple interactions of VDAC with molecular partners were described to occur around E73. It is conceivable that these mechanisms do not act independently of each other, but a complex interplay between these factors adapts VDAC function to the current state of the cell . In thisThis complex interplay of regulatory mechanisms allows VDACs to adapt to changes in the metabolic state of the cell mediated by multiple layers of regulation and fine-tuning. However, many unanswered questions within this network are still open and E73 still remains to be one of the big enigmas of VDAC.


Here we investigated the relationship between the increase in Ca2+ and ROS levels immediately after the exposure to misfolded protein oligomers, asking whether they are simultaneous or instead one precedes the other. Using Aβ42-derived diffusible ligands (ADDLs) and type A HypF-N model oligomers (OAs), we followed the kinetics of ROS production and Ca2+ influx in human neuroblastoma SH-SY5Y cells and rat primary cortical neurons in a variety of conditions. In all cases we found a faster increase of intracellular Ca2+ than ROS levels, and a lag phase in the latter process. A Ca2+-deprived cell medium prevented the increase of intracellular Ca2+ ions and abolished ROS production. By contrast, treatment with antioxidant agents prevented ROS formation, did not prevent the initial Ca2+ flux, but allowed the cells to react to the initial calcium dyshomeostasis, restoring later the normal levels of the ions. These results reveal a mechanism in which the entry of Ca2+ causes the production of ROS in cells challenged by aberrant protein oligomers.Alzheimer's disease is characterized by the accumulation in the brain of the amyloid β (Aβ) peptide in the form of senile plaques. According to the amyloid hypothesis, the aggregation process of Aβ also generates smaller soluble misfolded oligomers that contribute to disease progression. One of the mechanisms of Aβ oligomer cytotoxicity is the aberrant interaction of these species with the phospholipid bilayer of cell membranes, with a consequent increase in cytosolic CaThe online version contains supplementary material available at 10.1007/s00018-022-04513-w. In eceptors . Other C (TRPM2) , the vol (VDCCs) , and the (VDCCs) .2+ increase induced by the oligomers and the consequent increased need for ATP by the Ca2+ pumps, that try to restore the normal levels of intracellular Ca2+ [Another relevant early biochemical change resulting from the interaction of Aβ oligomers with cell membranes is oxidative stress, which is associated with the accumulation of ROS in the cytosol , 15, 16 lar Ca2+ , 28. It lar Ca2+ –31, or wlar Ca2+ . On the lar Ca2+ –31. Anallar Ca2+ . Moreovelar Ca2+ . Eventua2+ signalling induced by misfolded protein oligomers can be considered as bidirectional, or whether one of them is causative of the other. Oxidative stress has been considered, at least in part, a consequence of Ca2+ entry into cells, because of the increased need to produce ATP by mitochondria to pump out Ca2+ ions, which produces ROS itself [2+ homeostasis in the cell and intracellular Ca2+ stores by oxidising multiple methionine residues within the Ca2+ signalling protein calmodulin (CaM) resulting in an inability to activate a range of target proteins, including the cell membrane Ca-ATPase involved in the maintenance of Ca2+ homeostasis [2+ influx induced by misfolded protein oligomers were specifically inhibited, showing that the lack of an influx of Ca2+ ions into the cytosol can reduce the ROS production, whereas the protection against ROS formation did not prevent the initial Ca2+ flux but allowed the cells to react, on a longer term, to the initial Ca2+ dyshomeostasis, restoring the normal levels of the ions.It is not yet clear, however, if interactions and cause-and-effect relationships between ROS production and CaS itself , 28, 35.eostasis , 36, 37.E. coli was prepared and purified as described [v/v) trifluoroethanol (TFE), 2 mM DTT, pH 5.5, 25 °C, for 4 h without agitation, as previously described [Wild-type HypF-N from escribed , and stoescribed .42  was dissolved in HFIP to 1.0 mM and incubated for 1 h at room temperature to allow complete peptide monomerization. Aβ42-derived diffusible ligands (ADDLs) were prepared as described previously [2 and the dried protein was resuspended to 5 mM with DMSO and then diluted with F-12 HAM to 100 μM. The sample was then incubated at 4 °C for 24 h and centrifuged at 12,000g for 10 min to collect the supernatant.Lyophilised Aβeviously . In partN-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) and NaHCO3 (1:1) and supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine and 1% antibiotics, as reported [2 humidified atmosphere at 37 °C and grown until they reached 80% confluence for a maximum of 20 passages. Primary rat cortical neurons (Thermo Fisher Scientific) were plated in 24-well plate at the density of 200,000 cells per well and maintained in neuronal basal plus medium (Thermo Fisher Scientific) supplemented with GlutaMAX (Gibco) at the concentration of 0.5 mM and 2% (v/v) B-27 serum-free complement (Gibco), in a 5% CO2 humidified atmosphere at 37 °C. Every 4 days the medium was partially replaced with fresh one. All the experiments were performed 12–16 days after plating.Human SH-SY5Y neuroblastoma cells  were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), F-12 HAM with 25 mM reported . Cell cu42 ADDLs diluted in cellular medium at the monomer equivalent concentration of 1 µM for 5, 10, 15, 30, 60, 90, 120 and 180 min. In other sets of experiments, before the treatment with HypF-N OAs or Aβ42 ADDLs, SH-SY5Y cells were pre-treated for 1 h with 5 µM CNQX, or 10 µM memantine, or both. In other sets of experiments, cells were pre-treated with 2 µM l-α-lysophosphatidylcholine (LPC) for 2 h, with 30 µM Trolox for 1 h, or in a medium without Ca2+ for 1 h. In another set of experiments, the SH-SY5Y cells were treated for 10 min with 1 mM NMDA or 50 µM AMPA, with or without pre-treatment with 30 µM Trolox for 1 h.SH-SY5Y cells were plated in 6-well plates containing coverslips at a density of 40,000 cells per well at 37 °C. After 24 h, they were washed with PBS and treated with HypF-N OAs diluted in cellular medium at the monomer equivalent concentration of 12 µM for 5, 10, 15, 30 and 60 min, or with Aβ42 ADDLs diluted in cellular medium at the monomer equivalent concentration of 1 µM for 10 and 60 min. In other sets of experiments, before the treatment with Aβ42 ADDLs, the cells were pre-treated for 1 h with 5 µM CNQX, or 10 µM memantine, or 30 µM Trolox.Primary rat cortical neurons were plated in 24-well plates containing glass coverslips coated with poly-D-lysine at a density of 200,000 cells per well at 37 °C. 12–16 days after plating, they were washed with PBS and treated with Aβ2+ levels were measured in living SH-SY5Y cells and primary rat cortical neurons after the different treatments, or after adding 1 µM ionomycin for 1 h as a positive control. The cells were then washed with PBS and loaded with 4 µM Fluo-4 AM (Thermo Fisher Scientific) for 10 min. The Ca2+ levels for untreated cells were evaluated in SH-SY5Y cells at each time point, from 0 to 180 min, changing the cellular medium at the different time lengths, washing with PBS and loading the Fluo-4 AM probe for 10 min. Considering the absence of changes in basal Ca2+ levels, all data are reported relative to untreated cells at time 0. ROS levels were measured in living SH-SY5Y cells and primary rat cortical neurons after the different treatments, or after adding 250 µM H2O2 for 1 h, as a positive control, and then by loading 5 µM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) in the last 15 min of the different treatments. ROS levels for untreated cells were evaluated in SH-SY5Y cells at the different time points, changing the cell medium and loading the probe in the last 15 min of the treatment. Considering the absence of significant variation, all data are reported relative to untreated cells at 15 min, which is the probe incubation time and therefore the shortest time that can be analysed. Both Ca2+ and ROS levels were then evaluated after excitation at 488 nm by a TCS SP8 scanning confocal microscopy system equipped with an argon laser source (Leica Microsystems).Cytosolic Ca2+ and ROS levels were measured in living SH-SY5Y cells by loading 5 µM X-Rhod-1 AM (Thermo Fisher Scientific) in the last 20 min and 5 µM CellRox™ Deep Red Reagent (Thermo Fisher Scientific) in the last 30 min of the different treatments, respectively. Ca2+ and ROS levels were then evaluated after excitation at 561 and 633 nm, respectively, by the same TCS SP8 scanning confocal microscopy system described above.In another set of experiments, cytosolic Ca2+ and ROS analyses, using Image J software. Values were averaged over the 10–22 cells in each experiment and the mean and standard error of the mean (SEM) were determined from the averaged values of the three experiments (n = 3). All data were normalized to the positive control value, obtained with ionomycin and H2O2 respectively, which were attributed 100%.In all cases, a series of 1 µm thick optical sections (1024 × 1024) was taken through the cell depth for each sample using a Leica Plan Apo 63 × oil immersion objective and projected as a single composite image by superimposition (Leica Microsystems). Three different experiments were carried out and 10–22 cells were analysed in each experiment, in both Cap values < 0.05, < 0.01 and < 0.001, respectively.All data were expressed as means ± SEM (standard error of the mean). Comparisons between the different groups were performed by Student’s t-test. The single , double  and triple  symbols refer to 42 oligomers in cell and animal models [We started our analysis using model oligomers formed by the protein HypF-N , which were previously found to have effects similar to those of Aβl models , 41, 42.l models .42 ADDLs, are able to cause a progressive increase of intracellular Ca2+ levels on SH-SY5Y cells by activating rapidly extrasynaptic NMDA receptors and, to a lower extent, AMPA receptors [2+ levels, which was evident already after 5 min and reached the maximum level after 30 min of treatment  showed a slow and gradual increase of ROS production, which was slower than that observed by monitoring Ca2+ and ROS increases induced by HypF-N OAs in the absence of NMDA/AMPA inhibitors, the ROS time course appears to be slower, particularly in the first minutes  effectively inhibits the mechanical signal (stretching) generated by the action of the oligomers onto the membrane [2+ and ROS levels. In the presence of LPC, the Ca2+ levels remained similar to those of untreated cells up to 15 min; they then increased, but remained significantly lower than those recorded without LPC at corresponding time points, even after 60 min of treatment  inhibits the OA-induced Camembrane . To inveent Fig. a,b.Fig. 2+ flow through NMDA receptors for a prolonged time and the subsequent rise of ROS levels. They also confirm the presence of an interconnection between the two mechanisms.The ROS levels also remained constant for 15 min and then increased, but remained significantly lower than the corresponding values in the absence of LPC pre-treatment, up to 60 min Fig. c,d. Thes2+ is associated with the elevation of ROS following treatment with HypF-N OAs. The kinetic traces described so far lead to the hypothesis that the second event is caused by the first, rather than being independent of it, based on the observation that: (i) the former is more rapid than the latter, (ii) the rise of ROS has a lag time and follows the lag-independent rise of Ca2+, (iii) the lag times are longer in the ROS time courses than in the corresponding Ca2+ time courses in the presence of NMDA/AMPA inhibitors, and (iv) the effects caused by inhibitors of the Ca2+ flow (CNQX and memantine) are even larger on the time-dependent increase of ROS than Ca2+.As observed in the previous experiments, the intracellular rise of free Ca2+ concentration was observed in the early stages up 10 min, followed by a reduction down to levels observed in untreated cells .In a control experiment, to assess whether Trolox interferes directly with the AMPA and NMDA receptor opening, we activated the two receptors using their specific agonists, AMPA and NMDA, respectively, after a 1 h pre-treatment with 30 µM Trolox, finding that both agonists are able to induce an increase of the intracellular Ca2+ and ROS level increases, we treated the SH-SY5Y cells with HypF-N OAs over time, in a Ca2+-free medium .Taken together, the kinetic data obtained with Trolox and the Ca42-derived diffusible ligands (Aβ42 ADDLs) [42 ADDLs, similarly to HypF-N OAs, are able to cause a progressive increase of the intracellular Ca2+ levels in SH-SY5Y cells by activating rapidly extrasynaptic NMDA and AMPA receptors [42 ADDLs oligomers and evaluated whether they maintained this effect. The treatment over time of SH-SY5Y cells with Aβ42 ADDLs showed a gradual increase of the intracellular Ca2+ levels, which was clearly detectable already after 5 min and reached a plateau after 180 min of treatment   at the ceceptors . We ther42 ADDLs under the same conditions also showed a gradual increase of ROS production, which was evident after 15 min up to 180 min, and hence slower than that observed by monitoring Ca2+ concentration , and that ROS levels remained constant and similar to those of untreated cells when the treatment was performed in a medium without Ca2+ .To confirm these results with different probes of intracellular Ca42 ADDLs for 10 or 60 min, with or without the 1 h pre-treatment with Trolox. Similarly to SH-SY5Y cells, the presence of Trolox did not prevent a slight increase of the intracellular Ca2+ concentration, but caused lower levels of Ca2+ after both 10 and 60 min of treatment with Aβ42 ADDLs relative to cells pre-treated with Trolox  and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) need ATP to pump out the ions from the cytosol and restore homeostasis [2+ dyshomeostasis activates the Krebs cycle, electron transport chain and oxidative phosphorylation in mitochondria, which determines the mitochondrial generation of ROS through the increased O2 reduction [2+ [2+ dysregulation and increased ROS production.The maintenance of the Caeostasis , 36, 37.eduction , 37. ROS42 ADDLs and HypF-N OAs addition, is suggestive, albeit not a demonstration per se, that the ROS increase follows, and is caused by, that in Ca2+. To address further the cause-and-effect relationship between these two events, we took into consideration the data obtained with inhibitors of the Ca2+ influx and the known relationship between the two processes. Indeed, the extracellular-to-cytosol influx of Ca2+ induced by misfolded protein oligomers arises, at least in its early stages, from the passage of the ions through the AMPA and NMDA receptors [2+ influx induced by these oligomers, with no significant increase within the first minutes of treatment. The delay mediated by CNQX and memantine, however, did not only involve the Ca2+ influx, but also ROS production. We also observed a delay in ROS levels increase following the pre-treatment with the inhibitors and this delay was even larger than that observed for Ca2+ levels. These results suggest that the inhibition of AMPA and NMDA receptors, with the consequent reduction of the early Ca2+ influx, allowed the cells to postpone ROS production. At later time points, intracellular Ca2+ levels increase despite the persistent inactivation of the two receptors, reaching the same levels observed in the absence of any inhibition, because it is caused by the direct passage of the ions through the cell membrane after the interaction of the oligomers with the lipid bilayer and a consequent destabilization and perforation [2+ pumps are inhibited by ROS, contributing to increase Ca2+ levels at later time points (see below). In the same way, ROS production increases, while continuing to maintain this slight delay because of the inactivation of the AMPA/NMDA receptors. The kinetic data, in particular, indicate that the use of either CNQX or memantine, or both, results in a lag time of the increase in Ca2+ levels, followed by the extension of the lag phase in ROS production. Other Ca2+ channels are probably involved in the oligomer-mediated Ca2+ influx, such as TRPM2 [2+ and ROS kinetics was lower on cells treated with Aβ42 ADDLs, compared to cells treated with HypF-N OAs, probably because the damaging action of the first type of oligomer on the membrane is stronger, and, after its interaction with the lipid belayer and the consequent destabilization and perforation, the direct passage of the ions through the cell membrane is more pronounced compared to that of HypF-N OAs.The kinetic results presented here show that the delay in ROS production, which is evident as a lag phase and slower overall process in both time courses of ROS production following Aβeceptors , 18, 19,eceptors . The phaforation , 18. In as TRPM2 , VDCCs [as TRPM2  and TRPAas TRPM2 , but theas TRPM2 . The del2+ signalling protein calmodulin (CaM), with its consequent inability to activate its target proteins, including the PMCA, which is important for the maintenance of Ca2+ homeostasis [2+ homeostasis effectively, as a result of the lack of ROS-mediated oxidation of the PMCA and SERCA, among other cellular factors.In AD brains, high levels of intracellular ROS were found to react with several macromolecules, such as proteins, polysaccharides, nucleic acids and lipids, causing their oxidation and leading to the production of reactive ketone/aldehyde moieties and other carbonyl derivatives . An impoeostasis , 50, 51.eostasis –54. Inde2+ regulatory proteins mediated by ROS may represent an adaptive response to the oxidative stress, because it down-regulates ATP production through the mitochondrial electron transport chain and the inevitable generation of ROS associated with it [The selective oxidation and inactivation of the Ca with it .2+ influx in ROS production occurs in the treatment with the oligomers in a medium without Ca2+ (to inhibit Ca2+ influx) and with an antioxidant agent (to inhibit ROS production). The absence of Ca2+ in the extracellular medium fully inhibits the increase of the intracellular Ca2+ levels that normally flow from the extracellular space, but at the same time fully inhibits ROS production, confirming that ROS result from the need to restore Ca2+ homeostasis. By contrast, treatment with the antioxidant agent Trolox leads the restoration of Ca2+ homeostasis, but only at prolonged time points. This latter analysis showed that Ca2+ ions enter the cells in the first minutes, because the antioxidant agent inhibits only ROS production and is not able to inhibit the oligomer-mediated activation of AMPA and NMDA receptors that occurs within the first minutes of interaction of the oligomers with the cell membrane. This rapid increase of intracellular Ca2+ is followed by a decrease, suggesting that the cells are able to pump out Ca2+ and restore homeostasis as they benefit from an effective antioxidant capacity induced by Trolox and absence of any direct ROS-induced inhibition of the PMCA, SERCA, other pumps and possibly other cellular factors involved in these processes.Further evidence of the importance of Ca2+ signalling and ROS production [2+ signalling [2+ and cytosolic ROS production at the very early stages of the overall dysregulation induced by misfolded protein oligomers emerges from our results by three distinct lines of evidence, namely: (i) a lag time observed in the time course of oligomer-induced ROS production (but not in Ca2+ increase), (ii) an ability of AMPA/NMDA receptor inhibitors to retard ROS production even more effectively than Ca2+ influx and (iii) an inability of antioxidant agents to inhibit the early Ca2+ influx, while fully maintaining the redox status of the cells, whereas a Ca2+ deprived medium inhibits fully and effectively both Ca2+ influx and ROS production. Hence, the oligomers cause the entry of Ca2+ ions in the cells, determining the formation of ROS due to the increased demand of ROS-generating ATP production by mitochondria; ROS in turn prevent the cells from pumping back Ca2+ ions into the extracellular space and from restoring the normal Ca2+ homeostasis, indicating a positive feedback on Ca2+ dyshomeostasis on the longer time scale.Vicious cycles, or positive feedback loops, exist between Caoduction , 37, andgnalling  and betwgnalling , where tSupplementary file1 (DOCX 1182 KB)Below is the link to the electronic supplementary material.


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.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 Ca  Contractions of the muscle are resistant to tetrodotoxin, guanethidine, and atropine5,5. Atypical smooth muscle cells densely populate the pelvis–kidney junction (PKJ), where peristaltic contractions originate13. Recent evidence suggests that atypical smooth muscle cells possess fibroblast markers and express the Ca2+-activated Cl− channel (CaCC), Ano119. As a result of this finding, we now refer to atypical smooth muscle cells as platelet-derived growth factor receptor alpha-positive interstitial cells type 1 (PIC1). Changes in ANO1 expression is also implicated in disease. For example, in human ureteropelvic junction samples, decreased ANO1 expression is observed during ureteropelvic junction (UPJ) obstruction20. However, the mechanisms by which ANO1 channels participate in the peristaltic contractions of the RP are not fully understood.The process of actively transporting urine from the kidneys is vital. After ultrafiltration of the blood in the nephrons, concentrated urine flows into the upper urinary tract via the renal papilla. If urine is left to accumulate in the kidney, hydronephrosis can occur, characterized by a buildup of fluid and eventual renal failure. The renal pelvis (RP) is central to the process of exporting urine from the kidney. As urine enters the RP from the renal papilla, it flows through a connected series of minor and major calyces, and peristaltic contractions of smooth muscle cells that line the wall of the RP pump urine into the ureters toward the bladder7. Previous studies suggested that smooth muscle cells in the distal RP express tetraethylammonium and 4-aminopyridine sensitive K+ channels that affected the activation of L-type Ca2+ channels during propagating depolarizations24. It was also reported that the frequency of spontaneous electrical depolarizations is negatively correlated with the number of atypical smooth muscle cells along the RP from proximal to distal13. Several ion channels are proposed to be involved in propagating contractions, and expression analysis of murine RP showed that, in addition to Ano1, genes encoding voltage-gated Ca2+ channels, including L-type Ca2+ channels (CaV1.2 and CaV1.3) and T-type Ca2+ channels (CaV3.1 and CaV3.2), are expressed25. Other groups have suggested that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels initiate peristaltic contractions28, but few studies have attempted to understand the functional role of specific conductances in the contractile waves that propagate from the proximal RP to the ureter.Peristaltic contractions in the RP are initiated in typical smooth muscle cells in the proximal region and propagate distally for efficient transport of urine from the kidney2+ indicator, GCaMP3 in renal pelvic smooth muscle cells, making it possible to monitor Ca2+ transients that activate smooth muscle cells via voltage-dependent Ca2+ channels. This model makes it possible to visualize propagating peristaltic contractions, initiated at the PKJ and traversing the RP toward the ureter with high spatial and temporal resolution. We used this model to test our hypothesis that peristaltic contractions are initiated in the PKJ and propagate with relatively high efficiency to the distal pelvis and ureter and that the fidelity of propagation is provided by activation of specific voltage-dependent Ca2+ channel conductances and recruitment of ANO1 channels. The approach that we developed during this study allows accurate mapping of propagating Ca2+ waves underlying peristalsis. We found that T-type Ca2+ channels and the Ca2+-activated Cl− channel ANO1 are important determinants of peristaltic propagation distance in the RP.In the present study, we developed a model to monitor propagating peristaltic contractions in the RP. We utilized mice that conditionally express the genetically encoded Caad libitum, had free access to water, and were provided with appropriate enrichment. Mice were anesthetized by inhalation of 3%–4% isoflurane in oxygen. After induction of deep anesthesia  had been validated by loss of toe and/or tail pinch reflex, mice were euthanized by cervical dislocation.Mice were maintained and experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the Institutional Animal Care and Use Committee at the University of Nevada, Reno, NV, approved experimental protocols. Mice were fed GCaMP3lox/+ mice (B6.129S-tm38(CAG-GCaMP3)HzeGt(ROSA)26Sor/J) were crossed with male smMHC-iCre mice (B6.FVB-Tg Myh11-cre/ERT2) to generate smMHCGCaMP3/+ offspring (mice will be referred to as SMC-GCaMP). Both GCaMP3lox/+ and smMHC-iCre transgenic strains were purchased from The Jackson Laboratory. Since Cre recombinase expression is driven from the Y chromosome in B6.FVB-Tg Myh11-cre/ERT2 mice, only male mice underwent Cre recombination. Therefore, no female SMC-GCaMP3 mice were used in this study. Male SMC-GCaMP3 mice were used between 12 and 20 weeks of age. Male and female wild-type mice , aged 8–20 weeks, were used for contractile experiments.Female + cells by injection of tamoxifen into SMC-GCaMP3 mice at > 8 weeks of age (2 mg for three consecutive days). Tamoxifen (Sigma) was dissolved in ethanol  and vortexed for 20 min. Safflower oil was added, followed by sonication for 30 min to bring the final concentration of tamoxifen to 20 mg/mL. A total of 2 mg of tamoxifen was administered IP for three consecutive days for Cre recombinase induction. Successful induction was confirmed by genotyping 10 days after the initial tamoxifen injection. Once Cre recombinase expression had been confirmed, tissues from the mice were used for fluorescent imaging experiments.Expression of GCaMP3 was induced in smMHC3, 1.2 Na2HPO4, 1.2 MgCl2, 11.5 glucose, and 2.5 CaCl2, and bubbled with a mix of 97% O2 and 3% CO2. For SMC-GCaMP3 preparations, the RP was dissected from the surrounding parenchymal and adipose tissue. Care was taken during dissection to minimize trauma to the delicate PKJ. For wild-type contraction recordings, adipose tissue was sharply dissected from the distal RP, the renal capsule removed, and the kidney sliced sagittal with a blade to expose the PKJ and proximal RP. The papilla was then removed by cutting its connection with the cortex. An example of this preparation is shown in −1.Kidneys from SMC-GCaMP and wild-type mice were removed and immediately placed into ice-cold Krebs Ringer bicarbonate (Krebs) solution containing (in mM); 120.35 NaCl 5.9 KCl, 15.5 NaHCO29 software. After a control period, RP preparations were exposed to increasing concentrations of pharmacological compounds  for 10 min followed by a 5-min recording period. Vehicle control experiments were also performed in DMSO with a dose response application calculated from the highest concentration of DMSO used (0.01%).The distal RP and connected proximal ureter were imaged on an inverted Nikon Eclipse Ti microscope with a 2x Nikon objective. Images were acquired using a monochrome camera  and sampled at 20 Hz using IC Image Capture software (The Imaging Source). For online measurements of outer diameter (OD), images were sampled at 3 Hz using custom Vasotracker30 or Vasotracker software. For Fiji analysis, images were loaded in as AVI using the BioFormats plugin. A line-scan (50 px thickness) was drawn on the distal RP as it exits the kidney hilum and the reslice tool used to generate a spatio-temporal map of the tissue movement over time. The spatio-temporal map was thresholded to remove noise and a Gaussian filter was applied to smooth the trace (to improve maxima and minima peak detection). The entire spatio-temporal map was selected, and a plot profile of the spatio-temporal map was generated. The broadly applicable routines plugin package was used to find peaks (maxima and minima) in the plot profile. A list of detected peaks was generated that included the time point at which the maxima and minima occurred vs. peak height (amplitude). A custom Python script was used to automatically calculate the frequency, amplitude and peak–peak interval variance for each detected peak in the plot profile. Peak amplitudes below a specified threshold (mean–2 SD), were not counted as a detectable event for frequency, peak–peak interval or peak–peak interval variance calculations but were plotted for peak amplitude values. Diameter measurements were acquired using Vasotracker software (both online and offline versions). Multiple lines scan lines (10) were traced on to the RP using the ROI mode to measure the OD for each line  Ca2+ waves were visualized using an upright Nikon Eclipse E600FN microscope equipped with a 4x Plan Nikon objective lens. Images were acquired using an sCMOS camera . Images were sampled between 5 and 16 Hz using Andor Solis software. During image acquisition, the following protocol was used for each experiment: (1) control period 0–10 min, (2) pharmacological intervention period 10–30 min, and (3) washout period in Krebs solution 30–60 min. These time periods were chosen to allow time for drugs to reach specified bath concentrations during constant perfusion.Fluorescent Ca2+ waves in intact RP tissues were analyzed using spatio-temporal mapping. 8-bit time-lapse image stacks were converted to 32-bit image formats in Fiji software. For each raw image stack, the mean background intensity was sampled and subtracted from the entire series. To create spatio-temporal maps, line-scans were traced and extended through the entire RP, using either the straight- or segmented-line tool. The spatio-temporal map was then generated by evoking the reslice function. To calibrate spatio-temporal map intensity and generate F/F0 values, basal Ca2+ fluorescence (F0) was sampled and measured in a region of the pelvis during no activity . The entire time-series was then divided by F0 to provide a spatio-temporal map with amplitude expressed as F/F0.Propagation of Ca2+ waves (min−1) was calculated from these two regions.All representative traces for vehicle or pharmacological intervention experiments are derived from 1hour recordings or short-interval recordings of the intact RP at 5 Hz. The substack tool (in Fiji) was used to generate 2-min interval videos of the control period, drug incubation period, and washout period. Proximal renal pelvic SMC-GCaMP traces were generated from line-scans resliced in the proximal region of the pelvis, and distal renal pelvic SMC-GCaMP traces created from line-scans resliced from the distal region of the pelvis. The frequency of proximal and distal Ca2+ waves propagated along the RP, 2-min interval substacks from the entire time-lapse were resliced and spatio-temporal maps generated. Spatio-temporal maps were calibrated spatially and temporally. Using the straight-line tool, propagating Ca2+ waves were measured manually to derive a cumulative count for the total number of waves (frequency) and distance each Ca2+ wave traveled along the length of the entire RP (µm). Measurements were then saved as .txt files and analyzed. A custom Perl script was used to calculate the longest propagating Ca2+ wave across the pelvis during each recording. To normalize measurements, every other line from the spatio-temporal map was divided by the longest measurement within the spatio-temporal map.To measure the distance that Cainh-A01 (Tocris), benzbromarone (Tocris), nicardipine (Sigma), TTA-A2 (Alamone), and isradipine (Tocris) were dissolved in DMSO (concentration of DMSO in solution did not exceed 0.01%). ZD7288 (Tocris) was dissolved in water. Furosemide (Tocris) and bumetanide (Tocris) were dissolved in 100% ethanol (ethanol concentration did not exceed 0.01%). Nominal Ca2+ solutions (0 mM Ca2+ + 1 mM EGTA) contained (in mM): 120.35 NaCl 5.9 KCl, 15.5 NaHCO3, 1.2 Na2HPO4, 1.2 MgCl2, 11.5 glucose, 2.5 MgCl2, and 1.0 EGTA.CaCCt-test where appropriate. P-values < .05 are considered statistically significant. N refers to the number of animals used.All figures were plotted using R 4.1.3 (through RStudio) or GraphPad Prism (ver.9). All data are presented as means ± SD Statistical analyses were performed using Student’s paired or unpaired −1  and propagated down to the ureter . We observed a large range of Ca2+ wave propagation lengths in most preparations studied . Propagating Ca2+ waves traveled at a velocity of 624 ± 68 µm s−1 (N = 5). To assay spontaneous Ca2+ transient frequency, we selected regions of interest (ROIs) on the proximal and distal pelvis. The frequency of Ca2+ waves decreased between the proximal and distal regions .Coronal kidney sections with RP attached  and B we N = 37; . The frearations . Under ce ureter . Time-laver time . Lines w2+ waves  were mea regions . This re2, contractions occurred at regular intervals and tone was maintained , the RP lost tone . Example traces from the contraction assay performed on wild-type RP show minimal effects of DMSO even at the highest solvent concentration used . However, contraction amplitude was stable throughout the recording period but decreased significantly at 10−4% DMSO in solution but recovered thereafter (N = 4). Peak–peak interval variance did not change significantly as a function of DMSO concentration (N = 4), but RP mean diameter tended to fluctuate (N = 4). For SMC-GCaMP3 tissues, representative traces in 2+ wave propagation did not change significantly between control and DMSO application . Similarly, SMC-GCaMP3 wave frequency was not significantly different between control and DMSO  or distal  RP.The effects of DMSO on RP contractions were also tested. DMSO was the vehicle used for most pharmacological compounds studied , suggesting a relatively high degree of resistance toward this L-type Ca2+ channel antagonist  did not significantly affect normalized Ca2+ wave propagation distance . Example spatio-temporal maps for propagating Ca2+ waves are shown in P = .07, N = 5 for each group) or distal  RP when exposed to nicardipine (10−6 M). Example traces from a SMC-GCaMP3 preparation showed that whilst Ca2+ wave frequency remained constant, Ca2+ wave amplitude in the distal RP was reduced by nicardipine (10−6 M). These observations reinforce previous reports that the effects of dihydropyridines are highly variable in the murine RP7.As peristaltic contractions involve excitation–contraction coupling in smooth muscle cells, we investigated antagonists of Catagonist . Similarardipine . This waardipine . Contracardipine . Peak–petrations  and F. S−9 and 10−5 M) reduced contraction frequency  were not rhythmic. RP diameter significantly increased at 10−6–10−5 M isradipine, suggesting a loss of tone  and Ca2+ waves were completely absent in spatio-temporal maps (−6 M) also inhibited resolution of Ca2+ waves in the proximal  and distal  RP  RP . Example2+ channels to peristaltic contractions were also tested by exposing preparations to increasing doses of TTA-A2, a potent inhibitor of CaV3.1 and CaV3.2 channels33. At 10−6 M, TTA-A2, reduced contraction frequency in wild-type preparation and completely abolished contractions at 10−5 M  increased the peak-to-peak interval variance (−6 and 10−5 M (−6 M) significantly reduced the propagation distance of Ca2+ waves . An example spatio-temporal map in 2+ waves propagating toward the distal pelvis in the presence of 10−6 M TTA-A2. In addition to a reduction in propagating contractions, TTA-A2 significantly decreased the frequency of SMC-GCaMP3 Ca2+ waves in the proximal  and distal  RP  RP . Figure 18. Therefore, we tested the effects of two ANO1 antagonists, benzbromarone and CaCCInh-A01, on peristaltic contractions. Benzbromarone (10−5 M), abolished RP contractions . An example spatio-temporal map in 2+ waves failed to propagate down the length of the RP in the presence of benzbromarone (3 µM). This compound also reduced the frequency of Ca2+ waves in the proximal  and distal  RP  RP . Traces l pelvis .−5 M), a more selective inhibitor of CaCCs, reduced the frequency and amplitude of contractions significantly . A spatio-temporal map from one experiment shows a reduction in the number of Ca2+ waves propagating the entire length of the RP  and distal (P < .0001) RP in the presence of CaCCinh-A01 . CaCCInh-A01 (10−6 or 10−5 M) did not significantly reduce responses to 60 mM KCl solution . To further support a role for CaCCs in RP contraction propagation, we used two different Na+/K+/2Cl− cotransporter (NKCC) inhibitors, furosemide and bumetanide. NKCC is important for intracellular Cl− accumulation and, therefore, determines the outward Cl− gradient35. Although we tested both compounds, it is well-documented that bumetanide is more potent vs. furosemide36. In gastrointestinal interstitial cells of Cajal, use of bumetanide blocks CaCC spontaneous inward currents37. Since furosemide and bumetanide are both solubilized in ethanol, we first performed vehicle control experiments to determine effects, if any, of ethanol alone  contractions were rhythmic (−5 M) reduced the amplitude of Ca2+ waves in the distal RP and increased their frequency but had no effect on proximal Ca2+ wave frequency or amplitude . A spatio-temporal map from one experiment illustrates the increase in propagation length of Ca2+ waves in the presence of ZD7288 . However, the frequency of distal Ca2+ waves was increased significantly in the presence of ZD7288 , reflecting the increase in the length of Ca2+ wave propagation in the presence of ZD7288. Panels in 2+ waves do not change, whereas distal waves become more regular and frequent.A previous study reported that ZD7288 (30 µM), a pan-HCN antagonist, disrupts contractile regularity of the RP and eventually abolishes contractionsrhythmic . There wrhythmic , suggestmplitude . A total2+ waves , such th8 10 µM; . ZD7288 2+ waves along the length of the RP. We found that inhibition of voltage-gated Ca2+ channels had variable effects on Ca2+ wave frequency and propagation distance. Contractions and Ca2+ waves proved resistant to nicardipine, however, another L-type Ca2+ antagonist, isradipine, abolished propagating Ca2+ waves and contractions. Inhibiting T-type Ca2+ channels also reduced the frequency and propagation distance of Ca2+ waves. Notably, antagonists of ANO1 channels also decreased the propagation distance of Ca2+ waves and, in some cases, inhibited peristaltic propagation to the distal pelvis. Changing the chloride gradient pharmacologically with bumetanide decreased contraction frequency and caused irregular contractions. Rather than blocking generation and/or propagation of Ca2+ waves as previously reported26, an HCN channel antagonist increased the number of events propagating from proximal to distal pelvis .In this study, we utilized wild-type and transgenic SMC-GCaMP3 mice to elucidate the propagation mechanisms of pyeloureteric peristaltic contractions. We visualized contractile responses to increasing concentrations of several ion channel antagonists. To complement this approach and to understand mechanisms that underlie peristaltic wave generation and propagation, we monitored the movement of Ca2+ serve as the fundamental pacemaker signal in specialized interstitial cells, typically via activation of Ca2+-dependent ionic conductances10), pacemakers of the RP, are abundant in the PKJ where contractions initiate. PIC1s express ANO1 and exhibit spontaneous endoplasmic reticulum-mediated Ca2+ transients18. The Ca2+ transients likely couple to openings of ANO1 channels to produce spontaneous transient inward currents (STICs). STICs have been reported in isolated atypical smooth muscle cells and are thought to coalesce into large inward currents to trigger spontaneous transient depolarizations6. Spontaneous transient depolarizations are likely the depolarization event that activate voltage-gated Ca2+ channels, facilitating the propagation of peristaltic contractions from the PKJ to the ureter. Previous pharmacological assays investigating the role of CaCCs in the RP led to inconsistent results, possibly due to the use of nonspecific Cl− channel antagonists. For example, 4,4′‐diisothiocyano‐2,2′‐stilbenedisulfonic acid did not affect spontaneous transient depolarization frequency in murine proximal RP7. However, niflumic acid (NFA) reduced the frequency of events in guinea pig proximal RP45 and reduced STICs in atypical smooth muscle cells 6. Second generation ANO1 inhibitors, such as benzbromarone and CaCCinh-A01, have been used to evaluate the role of CaCCs in pacemaker activity in the gastrointestinal tract42, and urethra50. In the present study, we found that these antagonists had significant effects on the propagating Ca2+ waves that underlie RP contractions. Although we found that CaCCInh-A01 inhibition had no significant effects on KCl-induced contractions, this does not completely exclude possible inhibition of L-type Ca2+ channels. Even a small inhibition of L-type Ca2+ channels by CaCCInh-A01 could be masked in largely depolarized cells. Further studies should consider using isolated RP smooth muscle cells to determine effect on L-type Ca2+ channel conductance. Despite this caveat, in combination with furosemide and bumetanide experiments, these data suggest that CaCCs reinforce the propagation of peristaltic contractions, thereby increasing the safety factor for propagation from the proximal to distal regions. Since ANO1 channels are expressed in PICs, we speculate that these cells provide a means of boosting inward currents and facilitate active peristaltic propagation18.The mechanisms that initiate activation of pacemakers to drive smooth muscle cell peristalsis in the RP are not fully understood, but CaCCs may provide a means to initiate contractions. In some smooth muscle tissues, transient increases in intracellular Ca2+ channels in regulating RP peristalsis is also emerging. In other visceral organs and the sinoatrial node, L-type Ca2+ channels  and low-voltage-gated T-type Ca2+ channels are involved in pacemaker activity2+ channel expression has not been demonstrated immunohistochemically due to the lack of specific antibodies for channel subtypes, however, T-type Ca2+ channels expression has been determined in multiple species. T-type Ca2+ channels are present in murine PKJ60 and porcine and human calyces25. Previous studies suggest T-type expression is location dependent with higher expression of CaV3.1 prevailing in the PKJ and tapering off in the mid-distal RP27. Similar expression patterns also exist for the other CaV3 subtypes. Based on molecular expression data, CaV3.2 and CaV3.3 are more highly expressed in the distal vs. the proximal pelvis18. Collectively this suggests that T-type Ca2+ channels may be important in typical smooth muscle cells rather than PIC1s, although more cell-specific data is required to support this conclusion. The functional significance of T-type expression has also been previously investigated. T-type antagonists are known to reduce the frequency of pelvic contractions60. Although we also observe that T-type Ca2+ channels inhibition reduces contraction frequency, our SMC-GCaMP3 imaging data demonstrates that T-type Ca2+ channels are also required for ensuring proximal-to-distal peristaltic propagation . In our study, we found that TTA-A2, a potent, voltage-dependent, pan- T-type Ca2+ channels antagonist32 significantly reduced peristaltic transmission to the distal pelvis. We postulate that T-type Ca2+ channels expression in typical smooth muscle cells may promote propagation of Ca2+ waves associated with peristalsis. This is particularly important in the distal RP that has more negative resting potentials compared to the proximal region13. Together, T-type Ca2+ and CaCC channels may contribute toward providing more depolarized membrane potentials in the distal RP. This mechanism may be important once a propagating action potential reaches the distal portions of the RP. Due to increasing expression of 4-AP sensitive K+ channels in the distal RP, membrane potential is more hyperpolarized14. Therefore, we speculate that T-type Ca2+ channels and CaCCs will dictate if a wavefront passes through to the ureter.The role of voltage-gated Ca2+ channels inhibition had robust effects on contractions, similar to other reports, we found that dihydropyridines which antagonize LTCCs had variable effects on RP contractions60. One study demonstrated that nifedipine decreased RP contraction frequency, action potential discharge, and caused membrane depolarization7. However, in another study by the same group, nifedipine either totally abolished or had minimal effects on Ca2+ waves in Ca2+-dye-loaded pelvis preparations7. In our study, we found that high concentrations (up to 10−4 M) of nicardipine failed to elicit significant effects on contractile properties. During our SMC-GCaMP3 assays, we also found that nicardipine (10−6 M) did not reduce Ca2+ wave propagation or affected proximal or distal Ca2+ wave frequency. The negligible effects of nicardipine contrasted sharply with the rapid inhibitory effects of isradipine on Ca2+ waves. One possible explanation may be T-type Ca2+ channel affinity for nicardipine and isradipine. A study by Perez-Reyes and colleagues found that isradipine potently inhibits T-type Ca2+ channels with an IC50 of < 3 μM61. Our data suggests that concentrations between 1 and 10 μM are sufficient to significantly inhibit and abolish contractions. Another explanation for difference between nicardipine and isradipine effects may be attributed to the specific repertoire of CaV1 family channels expressed in the RP. Previous molecular characterization of the RP revealed that transcript levels of Cacna1d (encoding the α subunit of CaV1.3) are more abundant in the proximal and distal pelvis, as compared to expression of Cacna1c18 (encoding the α subunit of CaV1.2). The sensitivity of CaV1 subtypes to dihydropyridines in RP smooth muscle cells has not been extensively characterized, but CaV1.3 channels may contribute more substantially to excitation–contraction coupling in the pelvis than CaV1.2 channels. CaV1.3 channels activate at more negative potentials as compared to CaV1.2 channels62, so CaV1.3 may provide an additional safety factor to ensure that contractions initiated in the proximal RP propagate through to the ureter. This mechanism may initiate excitation-contraction coupling at lower levels of depolarization, ensuring that the excitatory events in proximal pacemaker cells propagate through to the distal pelvis. Future studies should attempt to determine whether CaV1.2 and/or CaV1.3 are important for excitation–contraction coupling in the RP. The contributions of CaV1.2 and CaV1.3 will need to be elucidated in studies where the dihydropyridine sensitivity of these channels has been genetically modified63.Although T-type Ca26 and multicalyceal species 25. ZD7288 is effective as an antagonist for HCN channels (IC50: 15 µM)64, but nonspecific effects on ICa and INa have also been reported64. For example, low concentrations of ZD7288 (< 1 µM) reduced ICa current sinoatrial nodal cells65, and higher concentrations (1–30 µM) reduced INa current in dorsal root ganglion neurons64. In our study, ZD7288 did not significantly affect contraction frequency or amplitude at low concentrations, and higher concentrations of ZD7288 (10 or 30 µM) potentiated the propagation of Ca2+ waves in the distal RP, such that the majority of Ca2+ waves propagated from the PKJ to the ureter. Thus, our observations differ significantly from the effects attributed to this compound in prior reports. Contrastingly, we did not observe uncoordinated peristalsis  at any ZD7288 concentration tested. A possible explanation for the differences in our observations and previous observations could be that we recorded propagating Ca2+ waves associated with peristalsis, whereas previous reports utilized voltage-sensitive dyes to monitor depolarizations in the RP. The voltage-sensitive dyes used in prior studies suffer from low sensitivity and resolution and could possibly have resulted in misinterpretations of the effects of the drugs. Voltage-sensitive dyes are best used for measuring large changes in membrane potential, which may not be a feature of many smooth muscle tissues. If ZD7288 affects the magnitude of ICa, which is likely to be a feature of the electrical activity of cells in the RP, then it may have been difficult to detect these events in the presence of this drug. We also speculate that ZD7288 may block K+ conductances since we observe an improvement in the number of Ca2+ waves associated with peristalsis traveling down to the ureter. Additional experiments using conditional knock down of HCN3 will be necessary to clarify the role of this channel in peristaltic contraction modulation.The pacemaker channel HCN3, has been proposed as a conductance responsible for coordinating and triggering pyeloureteric peristalsis66, our SMC-GCaMP3 assay provides greater spatial and temporal resolution to track propagating Ca2+ waves associated with contraction from the proximal RP through to the ureter. Our experiments have revealed novel and variable effects of L-type Ca2+ channel antagonists, nicardipine and isradipine, that seem likely due to different contributions of voltage-gated Ca2+ channels and effects of isradipine on T-type Ca2+ channels. We also revealed that T-type Ca2+ channels and CaCCs contribute to the safety factor for propagation of peristaltic contractions from the proximal pacemakers to the ureter. The integrated output of several inward current conductances is likely to be important for efficient transport of urine in the RP. Boluses of urine are carried to the ureter with each peristaltic wave. If the probability of bolus transport is reduced by incomplete peristalsis, this could negatively impact hydrostatic pressure in the renal papilla and the tubulointerstitium upstream, causing an overall loss of nephrons. Future studies using cell-specific genetic ablation of T-type Ca2+ channels and CaCCs will be important to acknowledge which cells and conductances provide the physiological safety factor for peristaltic contractions in the RP.This study demonstrates improved approaches for investigating the mechanisms of propagation of peristaltic contractions in the RP using two imaging modalities and analyses. These tools will be valuable as means of evaluating other pharmacological compounds, genetic interventions, and the consequences of pathophysiological challenges. Our preparations maintain coupling between the proximal and distal RP, a feature typically missing in muscle strip studies. However, whilst other studies have carried out experiments with the RP intactzqac041_Supplemental_VideoClick here for additional data file.


Gliosarcoma is a rare subtype of glioblastoma, isocitrate dehydrogenase (IDH) wildtype. This biphasic tumor has two components. The first one is glial and usually represented by glioblastoma. The second is a sarcomatous component usually represented by nonspecific spindle cell sarcoma. Rarely, different glial tumors could represent the non-sarcomatous component, including oligodendroglioma and ependymoma. There were only two reported cases in the literature with glioneuronal components (both were anaplastic ganglioglioma) as the non-sarcomatous component. This work reports a gliosarcoma in the right frontal lobe of a 13-year-old female with a glioneuronal tumor representing the non-sarcomatous component and a rhabdomyosarcoma representing the sarcomatous component. The child lived for only six months after the resection of the tumor. The short survival attests to the dismal prognosis of gliosarcoma regardless of the nature of the non-sarcomatous component. Gliosarcoma is a rare subtype of glioblastoma, isocitrate dehydrogenase (IDH) wildtype, and it is considered a central nervous system (CNS) World Health Organization (WHO) grade 4 tumor . It is cA 13-year-old female patient was diagnosed with T-cell acute lymphoblastic leukemia with CNS involvement at the age of six years. She was treated with combined chemotherapy and radiotherapy. She was presented to the emergency department with a progressive left-sided weakness for three weeks and headache and vomiting for one week. The past medical history was significant for primary hypothyroidism, growth hormone deficiency, and primary ovarian failure.Magnetic resonance imaging (MRI) examination of the brain showed a large (4.4 cm x 3.19 cm x 3.4 cm) right frontal mass with heterogeneous enhancement . The result was reported as no significant alteration was detected. The integrated diagnosis was gliosarcoma with glioneuronal component and rhabdomyosarcoma. The patient was on palliative treatment and passed away six months after resection.This case is the third reported gliosarcoma, up to my knowledge, with a glioneuronal tumor as the non-sarcomatous component. The non-sarcomatous component is difficult to classify in this case. The presence of infiltrative neoplastic astrocytes and dysmorphic ganglions indicated a glioneuronal tumor. Among glioneuronal tumors, anaplastic ganglioglioma would be the most likely possibility. The molecular test did not show evidence of mitogen-activated protein kinase (MAPK) pathway alteration. This alteration is an essential diagnostic criterion for an unresolved ganglioglioma . HoweverThere are only two previously reported cases in which the non-sarcomatous component represents a glioneuronal tumor. The glioneuronal component in these two cases was considered an anaplastic ganglioglioma. Suzuki et al. reported a case of anaplastic ganglioglioma with an undifferentiated sarcoma component in a 59-year-old male. The ganglioglioma contained a classic low-grade area with a transition to another area with anaplastic features. The spindle cells in the sarcomatous component were immunopositive for smooth muscle actin (SMA) but immunonegative for desmin and cluster of differentiation (CD) 34. The sarcomatous component was considered fibrosarcoma or malignant fibrous histiocytoma. The clinical follow-up of the patient was not documented [Zhi et al. reported another gliosarcoma case with anaplastic ganglioglioma in a 46-year-old male. The tumor was circumscribed solid and cystic in the left temporal lobe that extended to the optic chiasm and brainstem. The ganglioglioma component comprised dysmorphic ganglions and glioma with high-grade features . The sarcomatous component was formed by undifferentiated pleomorphic and spindle neoplastic cells and areas of pseudopalisading necroses. These cells were immunonegative for SMA, desmin, CD34, CD68, and epithelium membrane antigen (EMA) immunostains. Unfortunately, the follow-up was only three months after the surgery [This report is the third gliosarcoma case, to my knowledge, with a glioneuronal component representing the non-sarcomatous component and rhabdomyosarcoma representing the sarcomatous component. The most common glial component in gliosarcoma is glioblastoma (WHO grade 4), which could explain the poor prognosis in these tumors. However, in some cases, the prognosis is even worse than glioblastoma. The spectrum of the non-sarcomatous component is expanding to include tumors with lower WHO grades such as oligodendroglioma and ependymoma. This report expands the spectrum of these cases by confirming the possibility of a glioneuronal component (WHO grade 3) as the non-sarcomatous component. The bad prognosis, in this case, is similar to the prognosis of gliosarcoma with glioblastoma as the glial component. Future studies with more similar cases are required to confirm this bad prognosis.