Here we summarize the evidence from two “giant” presynaptic terminals—the squid giant synapse and the mammalian calyx of Held—supporting the involvement of nanodomain calcium signals in triggering of neurotransmitter release. At the squid synapse, there are three main lines of experimental evidence for nanodomain signaling. First, changing the size of the unitary calcium channel current by altering external calcium concentration causes a non-linear change in transmitter release, while changing the number of open channels by broadening the presynaptic action potential causes a linear change in release. Second, low-affinity calcium indicators, calcium chelators, and uncaging of calcium all suggest that presynaptic calcium concentrations are as high as hundreds of micromolar, which is more compatible with a nanodomain type of calcium signal. Finally, neurotransmitter release is much less affected by the slow calcium chelator, ethylene glycol tetraacetic acid (EGTA), in comparison to the rapid chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). Similarly, as the calyx of Held synapse matures, EGTA becomes less effective in attenuating transmitter release while the number of calcium channels required to trigger a single fusion event declines. This suggests a developmental transformation of microdomain to nanodomain coupling between calcium channels and transmitter release. Calcium imaging and uncaging experiments, in combination with simulations of calcium diffusion, indicate the peak calcium concentration seen by presynaptic calcium sensors reaches at least tens of micromolar at the calyx of Held. Taken together, data from these provide a compelling argument that nanodomain calcium signaling gates very rapid transmitter release. The first published model, by Chad and Eckert  while the channel is open, there will be an inverse relationship between Ca2+ concentration and distance from the channel; and (2) Ca2+ diffusion time will increase with the square of distance. To aid comprehension of these relationships, we provide “rule of thumb” Ca2+ diffusion parameters in Table D, as well as the unitary current flowing through the open Ca2+ channel and the properties of cellular Ca2+ buffering. More complete mathematical treatments can be found elsewhere, for example in Smith and Augustine  and in the right column of Table As mentioned above, it is possible that Cas Figure . This tys Kasai, . If therg Figure . Finallyr Figure . The difequation  and in t2+ signaling occur in the immediate vicinity of an open Ca2+ channel. The question addressed in this review is how relevant such domain signaling is for release of neurotransmitters from presynaptic terminals. We will compare Ca2+ signaling at two presynaptic terminals whose large size makes them particularly amenable to experimental analysis: the squid giant synapse and the mammalian calyx of Held. From results obtained at these synapses, as well as selected results from other synapses, we will develop the argument that nanodomain Ca2+ signaling is prevalent at presynaptic terminals that rapidly release neurotransmitters, though microdomain signaling also has been established for some types of presynaptic terminals. Radial gradients are likely to be important only for presynaptic terminals that slowly release neuropeptides or for slower modulatory presynaptic actions of Ca2+ within fast-acting synapses, for example Ca2+-dependent forms of synaptic plasticity.From a physical perspective, there is no question that local domains of Ca2+ domains in triggering neurotransmitter release were performed at the squid giant synapse. As a result, work at this synapse established most of the experimental paradigms that have been used to probe local Ca2+ signaling in presynaptic terminals. In this section we will describe some of these key experiments.Virtually all of the early experimental tests of the role of Ca2+ “tail” current  and the resultant postsynaptic response is very brief, on the order of hundreds of microseconds ethane-N,N,N’,N’-tetraacetic acid (BAPTA) were microinjected into the giant presynaptic terminal. A complex relationship between Ca2+ affinity and chelator efficacy was observed: the most potent inhibitor of transmitter release was dibromoBAPTA, a chelator with intermediate Ca2+ affinity (4.9 µM), while chelators with lower or higher affinities were less potent  binds Ca2+ more slowly because it is protonated at physiological pH and the bound proton must dissociate before Ca2+ can bind. This greatly slows the binding of Ca2+ to EGTA in comparison to BAPTA, even though the equilibrium affinity of these two chelators is roughly similar  filter for Ca2+.The final experimental paradigm—and the one that has proven most useful for probing local Ca2+ signaling processes, such as Ca2+ triggering of synaptic augmentation  to a highly digitated structure with stalks and swellings containing a total of 500–800 active zones at maturity  and excitatory postsynaptic currents (EPSCI) from young (P8–11) calyx of Held synapses, Borst and Sakmann  triggers fusion of a single synaptic vesicle. Furthermore, N-, P/Q, and R-type Ca2+ channels were found to jointly mediate transmitter release at this synapse, with P/Q-type Ca2+ channels being the most prominent subtype  the Ca2+ sensor  becomes more sensitive to Ca2+ to detect smaller rises in presynaptic Ca2+ concentration. To test the first possibility, Fedchyshyn and Wang , as previously reported  were significantly higher in immature synapses than in mature synapses. Based on the logic depicted in Figure 2+ channels required for triggering release of a synaptic vesicle decreases. It has also been shown that while transmitter release at P8–12 synapses requires both N- and P/Q-type Ca2+ channels, only P/Q-type Ca2+ channels are involved at mature synapses. This suggests that nanodomains contain specific type of Ca2+ channels as part of their molecular architecture.Two possible mechanisms have been considered: (1) the spatial coupling between open Caand Wang  injectedand Wang  further 2+ sensor sensitivity was measured in the developing calyx of Held synapse via simultaneous Ca2+ imaging and flash photolysis of caged Ca2+ chelators  in a spatially homogeneous manner. Surprisingly, Ca2+ sensor sensitivity decreases over development  while cooperativity (n) remains the same. Taken together, these studies led to the conclusion that the spatial coupling between Ca2+ channels and Ca2+ sensors switches from microdomain to nanodomain signaling.To test the second possibility, Ca2+ diffusion model and a five-site kinetic model of transmitter release (Naraghi and Neher, 2+ concentration seen by a Ca2+ sensor during an action potential increases from 35 to 56 µM (Figures 2+ channels and sensors; the modeling results indicate that the microdomain signaling during an action potential in immature synapses results from 12 Ca2+ channels with 50% open probability and 61 nm separation, while the nanodomain coupling at mature synapses results from 9 Ca2+ channels with 35% open probability and 23 nm spacing (Figure 2+ signaling nearly doubles the release rate and enables the peak of the local [Ca2+]i transient to occur ~410 µs earlier in mature synapses than in immature synapses (Figures 2+ channels at the developing calyx remains elusive, these simulation results provide a theoretical framework for envisaging how a developmental transition from microdomain to nanodomain coupling can recapitulate a number of experimental observations, including an explanation for how narrower action potentials become more effective in triggering transmitter release (Taschenberger and von Gersdorff, Such changes can account for the upregulation of transmitter release over the course of development of the calyx of Held synapse. Computer simulations with a linearized buffered Ca Figures . This isg Figure , 2009. T Figures . AlthougThe squid giant synapse and calyx of Held synapse are often considered to be specialized synapses because of their extraordinary size, speed and strength of synaptic transmission. Hence nanodomain coupling could be a feature unique to such high-fidelity, excitatory “relay” synapses. However, this clearly is not the case because a number of synapses have now been shown to release neurotransmitters via nanodomain coupling.2+ influx to transmitter release at the squid giant synapse and calyx of Held, early studies indicated that Ca2+ channels are tightly coupled to Ca2+ sensors to enable highly efficient transmission at neuromuscular synapses (Yoshikami et al., 2+ channel is sufficient for triggering of fusion events at the active zones of chick ciliary ganglion synapses (Stanley, In line with experiments suggesting nanodomain coupling of CaStanley, .2+ imaging in presynaptic terminals with patch-clamp recordings from postsynaptic neurons and treatment with subtype-specific Ca2+ channel blockers. The results collectively showed that not only are multiple Ca2+ channels involved in presynaptic Ca2+ signaling but also that different subtypes of Ca2+ channels can mediate transmitter release at a synapse, leading to the idea that opening of many adjacent Ca2+ channels forms microdomains.Initial studies in mammalian synapses appeared to argue in favor of microdomain coupling (Luebke et al., 2+ channels activated by an action potential differ, ranging from ~10% at the squid giant synapse to >70% at some mammalian central synapses. In addition, extracellular Ca2+ concentrations ranged from 11 mM for the squid synapse to 1–2 mM for mammalian central synapses and experimental temperatures varied from 15 to 37°C. All of these variables profoundly influence the number of Ca2+ channels opened by an action potential, as well as the driving force on Ca2+ and the kinetics of Ca2+ diffusion within nerve terminals, thereby affecting the coupling of Ca2+ channels to transmitter release.It should be noted that the conditions employed in these different experiments differ in many ways. For example, the fraction of Ca2+ signaling (Eggermann et al., 2+ signaling is found both at hippocampal basket cell-granule cell inhibitory synapses and at cerebellar parallel fiber-Purkinje cell excitatory synapses (Bucurenciu et al., 2+ triggering of rapid neurotransmitter release.Under experimental conditions where these variables have been somewhat controlled and are comparable to conditions employed at the developing calyx of Held synapse, emerging evidence suggests that several conventional mammalian central synapses employ nanodomain Ca2+ signaling that couples Ca2+ influx through presynaptic Ca2+ channels to vesicular fusion and synaptic transmission. There is no doubt that nanodomains exist at many synapses, but it is interesting to note that this mode of Ca2+ signaling appears to be primarily associated with synapses for fast-spiking neurons, where synaptic transmission is particularly important for preserving temporal precision of information flow (Yang et al., 2+ signaling may be particularly suited to trigger highly synchronized fusion events with minimal synaptic delay, temporal jitter, or asynchronous transmitter release. The narrow action potentials typical of fast-spiking neurons are likely to produce brief Ca2+ signals with rapid rise and collapse of local [Ca2+]. Brief Ca2+ transients will also serve to prevent presynaptic Ca2+ overload and facilitate Ca2+ clearance during prolonged high-frequency transmission. On the other hand, microdomains with loose coupling between Ca2+ channels and Ca2+ sensors may allow a broader dynamic range for tuning quantal output at synapses where activity-dependent forms of synaptic plasticity play a key role in dynamically regulating synaptic efficacy (Vyleta and Jonas, We have highlighted key evidence from two giant synapses, the squid giant synapse and the calyx of Held synapse, that firmly establish nanodomains as the predominant mode of Ca2+ signaling requires specific molecular specializations to optimize physical interactions between Ca2+ channels and SV (Catterall and Few, 2+ signaling in presynaptic terminals, it will next be important to define the specific molecular specializations underlying both microdomain and nanodomain Ca2+ signaling, and how these two modes of Ca2+ signaling are regulated during development. Emerging evidence indicate that several presynaptic proteins help tether Ca2+ channels to SV; these proteins include RIM (Kiyonaka et al., 2+ channels and sensors at Drosophila presynaptic terminals (Kittel et al., 2+ signaling are differentiated by specific molecular substrates and potentially could be inter-convertible to confer distinctive Ca2+ signaling properties to synapses.Given the dimensions of SV and active zones, it is reasonable to postulate that the nanodomain CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In RTAs from unoperated rats, these channels play a minor role in contractions and Bay K8644 (L-type channel agonist) mimics the effects of SCI. Here we investigated the mechanisms underlying the facilitatory actions of SCI and Bay K8644 on nerve-evoked contractions of RTAs and the hypothesis that Ca2+ entering via L-type Ca2+ channels is rapidly sequestered by the sarcoplasmic reticulum (SR) limiting its role in contraction. In situ electrochemical detection of noradrenaline was used to assess if Bay K8644 increased noradrenaline release. Perforated patch recordings were used to assess if SCI changed the Ca2+ current recorded in RTA myocytes. Wire myography was used to assess if SCI modified the effects of Bay K8644 and of interrupting SR Ca2+ uptake on nerve-evoked contractions. Bay K8644 did not change noradrenaline-induced oxidation currents. Neither the size nor gating of Ca2+ currents differed between myocytes from sham-operated (control) and SCI rats. Bay K8644 increased nerve-evoked contractions in RTAs from both control and SCI rats, but the magnitude of this effect was reduced by SCI. By contrast, depleting SR Ca2+ stores with ryanodine or cyclopiazonic acid selectively increased nerve-evoked contractions in control RTAs. Cyclopiazonic acid also selectively increased the blockade of these responses by nifedipine (L-type channel blocker) in control RTAs, whereas ryanodine increased the blockade produced by nifedipine in both groups of RTAs. These findings suggest that Ca2+ entering via L-type channels is normally rapidly sequestered limiting its access to the contractile mechanism. Furthermore, the findings suggest SCI reduces the role of this mechanism.In rat tail artery (RTA), spinal cord injury (SCI) increases nerve-evoked contractions and the contribution of L-type Ca In spinal cord injured (SCI) patients with high thoracic or cervical lesions, spinal reflex-evoked vasoconstriction elicited by distension of visceral organs, or by some other unheeded afferent input from caudal to the lesion, is exaggerated leading to large increases in blood pressure (autonomic dysreflexia (AD); 2+ channel blocker nifedipine 2+ entry via L-type Ca2+ channels normally plays a relatively minor role in nerve-evoked contractions and that the enhancement of nerve-evoked contractions produced by the L-type Ca2+ channel agonist Bay K8644 mimics that produced by SCI 2+ influx via L-type Ca2+ channels to contraction, but the mechanisms underlying this enhancement have not been determined.We have demonstrated that SCI markedly increases nerve-evoked constriction of rat tail artery and that this change is associated with an increase in sensitivity of these responses to inhibition by the L-type Ca2+ entering through N-type Ca2+ channels and does not normally involve Ca2+ influx through L-type Ca2+ channels 2+ channels Investigations of noradrenaline (NA) release from sympathetic nerve terminals in a range of tissues, including the rat tail artery, indicate that it is dependent primarily on Ca2+ channels, whose activity is increased by Bay K8644, normally play a relatively minor role in nerve-evoked contractions of the tail artery. Previous studies in tail artery have suggested that Ca2+ entering through L-type Ca2+ channels is rapidly sequestered by the sarcoplasmic reticulum (SR), limiting its access to the contractile machinery 2+ influx may overcome this Ca2+ buffering role of the SR, increasing access of Ca2+ entering via L-type Ca2+ channels to the contractile mechanism.The question remains why L-type Ca2+ sequestration by the SR increases the contribution of L-type Ca2+ channels to these responses. The findings demonstrate that SCI does not change the size or gating of Ca2+ channel currents recorded in tail artery myocytes. In addition, the findings demonstrate that depleting the SR Ca2+ store with ryanodine or interrupting Ca2+ uptake into the SR with the SR Ca2+-ATPase (SERCA) inhibitor cyclopiazoinc acid selectively increases nerve-evoked contractions in tail arteries from sham-operated rats and also increases the sensitivity of these responses to nifedipine.In the present study we investigated the mechanisms by which SCI and Bay K8644 increase nerve-evoked contractions and the possibility that reducing CaAll experimental procedures were approved by the University of Melbourne Animal Experimentation Ethics Committee and they conformed to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.−1 buprenorphine; Reckitt Benckiser, Sydney, Australia) by subcutaneous injection. Additional injections of saline and analgesic were administered daily for the first 3 post-operative days. Bladders were manually expressed 3 times daily until the animals regained the ability to empty their own bladders. In 17 age-matched sham-operated female rats, the laminectomy was performed to expose the spinal cord and post-operative treatments were similar except for bladder management. SCI and sham-operated rats were maintained for six weeks post surgery.The spinal cord was transected in 17 female Sprague Dawley rats  that were anaesthetized with isoflurane (2–3% in oxygen). A full description of the surgery is described in Al Dera et al +, 150.6; K+, 4.7; Ca2+, 2; Mg2+, 1.2; Cl−, 144.1; H2PO4−, 1.3; HCO3−, 16.3; glucose, 7.8. This solution was gassed with 95% O2 - 5% CO2 and warmed to 36–37°C.Rats were deeply anaesthetized with the volatile anesthetic isoflurane and killed by decapitation. The tail artery was dissected from 20–40 mm distal to the base of the tail. After isolation, arteries were maintained in physiological saline with the following composition (in mmol/L): Na50 or maximum contraction of tail arteries to phenylephrine Segments of artery (∼1.5 mm long) were mounted isometrically between stainless steel wires (50 µm diameter) in two 4-chamber myographs  as previously described The perivascular nerves were electrically stimulated with trains of 100 pulses  at 1 Hz applied through platinum plate electrodes mounted either side of the tissue. In preliminary experiments, it was confirmed that these stimulus parameters were supramaximal for activating the perivascular sympathetic axons and that contractions produced by electrical stimulation were fully blocked by tetrodotoxin (0.5 µM), confirming they are mediated by action potential-evoked neurotransmitter release. In all experiments assessing the effects of drugs on nerve-evoked contractions, the effects of each added drug was assessed 25 mins after its addition.−1 with physiological saline. The proximal end of the artery was drawn into a suction stimulating electrode and the perivascular nerves were electrically activated . Recordings were made at a site 1–2 mm distal to the mouth of the suction electrode with a carbon fibre electrode (7 µm diameter) that was mounted so that the first 100–200 µm from the tip of the fibre was in contact with the adventitial surface of the artery. The electrode was connected to an AMU130 Nanoamperometer  and a potential difference of +0.3 V was applied between the recording electrode and an Ag/AgCl pellet placed in the recording chamber medium. The current required to maintain this voltage was monitored.The release of endogenous NA was monitored using continuous amperometry in tail arteries from unoperated rats as described previously α1-adrenoceptor antagonist, prazosin (0.1 µM), to inhibit contractions due to released NA and the neuronal NA transporter inhibitor, desmethylimipramine (0.1 µM), to inhibit neuronal reuptake of released NA. During the experiments, the arteries were stimulated at 1 min intervals with 10 pulses at 10 Hz and Bay K8644 (0.1 µM) or cyclopiazonic acid  was added to the superfusing solution following the 10th train of stimuli and left in contact with the artery for a further 20 trains. At the end of the experiments, the Ca2+ channel blocker, Cd2+ (0.1 mM), was added to verify that the signals recorded were due to Ca2+-dependent release of NA 2/S1 ratios were also determined in tissues treated with the α2-adrenoceptor antagonist, idazoxan (0.1 µM), which increases NA release in the rat tail artery by interrupting α2-adenoceptor-mediated autoinhibition In these experiments, the physiological saline contained the 2+ free; Sigma-Aldrich, Castle Hill, NSW, Australia). The segments were incubated in dispersion solution (Hanks buffer (Ca2+ free) containing 0.7 mg ml−1 collagenase Type IA (Sigma-Aldrich), 0.5 mg ml−1 protease Type XIV (Sigma-Aldrich) and 2 mg ml−1 bovine serum albumin (Sigma-Aldrich)) for 10–14 min at 37°C in a 5% CO2 incubator. The tissue was then rinsed 4 times using Hanks buffer containing 2 mg ml−1 BSA. Smooth muscle cells (SMCs) were dispersed by gentle trituration of the segments with a wide-tipped fire-polished Pasteur pipette. The cell suspension was stored in Hanks buffer containing bovine serum albumin (2 mg ml−1) and Ca2+ (0.1 mM) at 4°C and used within 8 h.After dissection, tail arteries were cut open lengthwise and divided into small segments (∼2 mm in length) in Hanks buffer (Ca2+ currents (IBa) in myocytes were measured using the perforated patch configuration. The bath solution used to record IBa was composed of (in mmol/L) NaCl 130, KCl 5.4, BaCl2 10, glucose 10, EGTA 0.1 and HEPES 10 (pH adjusted to 7.4 with NaOH). The pipette solution for perforated patch recording contained (in mmol/L) CsMeSO4 120, tetraethylammonium Cl 20, EGTA 1, and HEPES 20 (pH adjusted to 7.2 with CsOH). Nystatin (10 mg/mL) was dissolved in dimethylsulfoxide (DMSO), sonicated, and diluted to give a final concentration of 150 µg/mL in the pipette solution. Inward currents were measured at room temperature using an Axopatch-1D patch-clamp amplifier, digitized with a 16-bit analog to digital converter , and controlled by pClamp8 (Axon Instruments).Bath and 100th pulse. As electrical stimulation produced an artefact  that lasted up to 500 ms following the last stimulus in the train, the amplitude of the NA-induced oxidation currents were measured 1 s following the last stimulus. The mean amplitudes of the three responses immediately before (S1) and 17–20 mins following the addition of Bay K8644 or CPZ (S2) were determined. The S2/S1 ratios in Bay K8644 or CPZ treated tissues were compared with those determined at the same points in control experiments with no drug added.The output from the myograph and amperometer was recorded and analyzed using a PowerLab data acquisition system and the program Chart . Because the contractions to 100 pulses at 1 Hz have both an initial phasic and a later tonic component, the amplitude of these responses was measured at both the 102+ channels were determined by calculating the peak conductance at each test potential by using the equation IBa = gBa×(V–Erev), where gBa, V, and Erev are peak conductance, test potential, and reversal potential, respectively. A double-pulse protocol was used to measure inactivation of Ca2+ channel current as a function of membrane potential. Conditioning steps from −80 to 30 mV  were applied for 3.5 s. After a 25 ms step to −80 mV, the membrane potential was stepped to 0 mV for 250 ms. Resulting currents were normalized to the maximum current obtained after a conditioning potential of −80 mV (I/Imax) and plotted as a function of the conditioning potential. The data were fitted by the Boltzmann equation using GraphPad Prism .To construct I–V relationships, cells were voltage-clamped at −80 mV and then stepped from −80 mV to +60 mV in 10 mV increments evoked 20 seconds apart. Activation relationships for the Cat-tests. When multiple effects were assessed in a single experiment, the data was first subjected to repeated measures ANOVA and the P values for pairwise comparisons were adjusted using the false discovery rate procedure P values <0.05 were considered to indicate significant differences. Unless otherwise indicated in the text, P values were obtained using unpaired t-tests. In all cases, n indicates the number of animals studied.All statistical comparisons were made using SPSS 22 .Bay K8644, nifedipine, phenylephrine HCl, prazosin, desmethylimipramine, carbachol, cyclopiazonic acid and nystatin were supplied by Sigma Aldrich . Ryanodine was supplied by Tocris Bioscience . Drugs were prepared as stock solutions in water , ethanol  or DMSO . In all experiments, the highest concentration of ethanol or DMSO applied was 0.1% (w vP<0.01). As a consequence the basal wall tension was larger for control arteries (4.3±0.2 mN mm−1) than it was for SCI arteries . These effects of SCI are similar to those previously reported −1; SCI: 7.7±0.7 mN mm−1; P = 0.29). Furthermore, when contracted with phenylephrine (3 µM), the % relaxation produced when the endothelium was stimulated with carbachol (1 µM) did not differ between control (47±7%) and SCI arteries .Mechanical responses were recorded in artery segments from twelve sham-operated rats (control arteries) and twelve SCI rats (SCI arteries). The estimated lumen diameter of the artery segments after the normalization procedure  was significantly smaller for the SCI arteries  on contractions evoked by 100 pulses at 1 Hz in control (n = 6) and SCI (n = 6) arteries. As previously reported ;P<0.01). In both groups of vessels, Bay K8644 increased the amplitude of contractions to this stimulus  than SCI arteries . We have previously reported that the facilitatory effect of Bay K8644 on nerve-evoked contractions is completely blocked by nifedipine 2+ channel activity. The finding that the facilitatory effect of Bay K8644 was much greater in control than SCI arteries may indicate that Ca2+ channel activity is elevated prior to adding this agent in SCI arteries.Using tail arteries from unoperated rat we have previously reported that the L-type CaIn situ amperometry was used to assess the possibility that Bay K8644 (0.1 µM) augments nerve-evoked contraction by increasing NA release from the sympathetic nerve terminals. These experiments used tail artery segments from unoperated rats that were stimulated with trains of 10 pulses at 10 Hz. The stimulation artifacts during the periods of stimulation prevented NA-induced oxidation currents evoked by individual stimuli being discerned, but summation of these signals produced a slowly decaying oxidation current following the last stimulus in the train (n = 6), Bay K8644 (n = 6) did not change NA release  whereas blockade of prejunctional α2-adrenoceptors with idazoxan  significantly increased the NA release . Membrane capacitance also was similar . Voltage pulse protocols were used to obtain the activation and inactivation curves for control and SCI SMCs (1/2) current activation  and inactivation  in control and SCI SMCs. In addition, in both groups of SMCs, application of Bay K8644 (1 µM) approximately doubled the amplitude of Ba2+ currents measured at 10 mV  and by inhibiting Ca2+ uptake into the SR with the SERCA inhibitor CPZ (1 µM). In these experiments, the tissues were stimulated with trains of 100 pulses at 1 Hz. In control arteries, both ryanodine (n = 6) and CPZ (n = 6) did not change the size of contractions measured at the 10th pulse during the trains of stimuli (th pulse (n = 6), where neither agent changed the size of nerve-evoked contractions . Importantly, in the presence of either ryanodine or CPZ, the size of the contractions measured at the 100th pulse did not differ significantly between control and SCI arteries . Because both ryanodine and CPZ selectively increased the size of nerve-evoked contractions in control arteries, these findings suggest that reduced Ca2+ sequestration by the SR contributes to the augmentation nerve-evoked contractions in SCI arteries.The possibility that a reduction in Cain situ amperometry. In comparison with S2/S1 ratios for NA-induced oxidation currents in time-dependent controls , those in tissues that were treated with CPZ did not differ significantly . Therefore a prejunctional site of action for ryanodine and CPZ can be excluded in the present study.A possible alternative explanation for the increase in nerve-evoked contraction produced by ryanodine and CPZ is if they act at a prejunctional site to increase neurotransmitter release. Previously it has been reported that 10 µM ryanodine does not change NA release in the rat tail artery 2+ sequestration by the SR limits the contribution of Ca2+ entering via L-type Ca2+ channels to nerve-evoked contraction, the effects of nifedipine (1 µM) were investigated in absence and in the presence of either ryanodine (10 µM) or CPZ (1 µM). In the absence of these agents, the % blockade of contractions produced by nifedipine at 10th pulse during trains of stimuli at 1 Hz was similar in control and SCI arteries (P = 0.48). By contrast, at the 100th pulse, the % blockade produced by nifedipine was greater in SCI arteries than in control arteries (P<0.05). The absolute size of the nifedipine-sensitive component of contraction at the 100th pulse was also larger in SCI arteries  and SCI (n = 6) arteries, the % blockade of nerve-evoked contractions produced by nifedipine was increased at both the 10th and 100th pulse . At the 10th pulse in the presence of ryanodine, the absolute size of the nifedipine-sensitive component of contraction was larger in SCI arteries than in control arteries  and SCI arteries (n = 6; th pulse, CPZ selectively increased the % blockade of contractions by nifedipine in control arteries (,2+ buffering by the SR increases the contribution of Ca2+ influx via L-type Ca2+ channels to nerve-evoked contractions.CPZ did not significantly change the % blockade of contractions produced by nifedipine at the 10s n = 6; Fig. 5D.2+ channels in SMCs. Furthermore, the greater augmentation of nerve-evoked contractions produced by Bay K8644 in control arteries does not appear to be explained by a greater stimulatory effect of this agent on L-type Ca2+ channel activity. Instead, the selective augmentation of nerve-evoked contractions produced by ryanodine and CPZ in control arteries, together with their greater sensitivity to nifedipine in the presence of these agents, suggests that Ca2+ entering via L-type Ca2+ channels is normally rapidly sequestered into the SR limiting its access to the contractile mechanism. As neither ryanodine nor CPZ increased nerve-evoked contractions of SCI arteries, we assume that Ca2+ entering via L-type Ca2+ channels is less susceptible to sequestration by the SR in these vessels and more freely accesses the contractile mechanism.The primary finding of this study is that the increased sensitivity of nerve-evoked contractions of tail arteries from SCI rats to nifedipine is not explained by an increase in functional membrane expression of L-type CaBay K8644 did not change the amplitude of nerve-evoked NA-induced oxidation currents, which provide a real time measure of NA release 2+ current carried by voltage-activated Ca2+ channels did not differ between SMCs from control and SCI tail arteries. There was also no difference in the voltage for half-maximal activation and inactivation of the Ba2+ current between the two groups of SMCs. When depolarized from −80 mV most of the Ba2+ current in tail artery SMCs is carried by L-type Ca2+ channels but there is also a component that is carried by T-type Ca2+ channels 2+ channel current in rat tail artery 2+ current produced by BayK8644 was similar, consistent with there being a similar contribution of L-type Ca2+ channel activation to the increase in Ca2+ conductance. Together these findings indicate that increased L-type Ca2+ channel expression in SMCs is unlikely to explain the greater contribution of these ion channels to neural activation of SCI tail arteries. The findings also indicate that the greater augmentation of nerve-evoked contractions produced by Bay K8644 in control arteries, compared to SCI arteries, is not explained by greater stimulation of L-type Ca2+ channel activity in these vessels.The size of the whole cell Bath pulse, both ryanodine and CPZ also produced a greater increase the % blockade of nerve-evoked contractions by nifedipine in control arteries. As a consequence, while in the absence of these agents the absolute size of the nifedipine-sensitive component of contraction at the 100th pulse was much larger in SCI arteries than in control arteries, in their presence it did not differ between these groups of vessels. These findings indicate that impairing the ability of the SR to accumulate and/or release Ca2+ increases the contribution of L-type Ca2+ channels to nerve-evoked contractions. Furthermore, as ryanodine and CPZ more markedly increased the contribution of L-type Ca2+ channels to neural activation of control arteries, this suggests that in these vessels Ca2+ entering via L-type Ca2+ channels is more susceptible to regulation by the SR than in SCI arteries.In control arteries, but not SCI arteries, both ryanodine and CPZ increased the size of nerve-evoked contractions measured at the end of the trains of 100 pulses at 1 Hz. As Bay K8644 increased the size of contractions in SCI arteries, the failure of ryanodine and CPA to increase these responses cannot be attributed to the muscle already being maximally activated. At the 100It has previously been demonstrated that ryanodine does not change NA release in the tail artery 2+ from the SR by locking the ryanodine receptor channels open 2+-induced Ca2+ release to activation of vascular muscle. In addition, the leak of Ca2+ from the SR induced by ryanodine prevents it from storing any Ca2+ accumulated by SERCA th pulse, ryanodine selectively and similarly increased the % blockade of contraction produced by nifedipine in both control and SCI arteries. Furthermore in the presence of ryanodine, the small nifedipine-resistant contractions measured at the 10th pulse did not differ between the groups of arteries. This selective action of ryanodine at the 10th pulse suggests that Ca2+-induced Ca2+ release contributes to the early phase of contraction in the rail artery. A similar suggestion has been made in rat mesenteric arteries where the early phasic contraction to long trains of nerve stimuli was inhibited by ryanodine but this agent did not affect the later tonic phase of contraction Ryanodine at 10 µM depletes Ca2+ influx via L-type Ca2+ channels to activation of tail artery smooth muscle 2+ entering the cell via L-type Ca2+ channels is rapidly sequestered into the SR limiting its access to the contractile machinery (see also 2+ channels to contraction following depletion of the intracellular Ca2+ stores has also been demonstrated in rat mesenteric veins 2+ chelation with BAPTA increased L-type Ca2+ channel activity recorded in cell attached patches. Therefore Thakali et al. 2+ released from the intracellular stores normally inactivates L-type Ca2+ channels. This process of inactivation is mediated via the Ca2+ sensor protein calmodulin, which binds to the intracellular C terminus of the Cav1.2 α1-subunit to cause a conformational change that enables channel inactivation th pulse in both control and SCI arteries indicates that Ca2+ released from a ryanodine-sensitive store normally inhibits L-type Ca2+ channel activity at this time point.Previously it has been reported that both ryanodine and CPZ increase the contribution of Casee also . As CPZ 2+ in vascular muscle cells are unknown. In rat vas deferens, the increase in smooth muscle reactivity produced by denervation is associated with a reduction in both SERCA Ca2+ uptake activity and protein expression of SERCA2 2+ homeostasis.The effects of SCI on sympathetic nerve-mediated activation of the tail artery are similar to those produced when the postganglionic sympathetic neurons supplying this vessel are decentralized by lesioning their preganglionic inputs 2+ buffering by the SR in control arteries suggests that Ca2+ entering via L-type Ca2+ channels is normally rapidly sequestered, reducing its access to the contractile mechanism (2+ buffering did not increase the size of these responses, it appears that sequestration of Ca2+ entering via L-type Ca2+ channels is lower in these vessels (2+ influx overcoming the Ca2+ buffering role of the SR (2+ released from intracellular stores inhibits the activity of L-type Ca2+ channels and that this is prevented by impairing ability of the SR to accumulate and release Ca2+In conclusion, the increase in both nerve-evoked contraction amplitude and sensitivity to nifedipine produced by disrupting cytoplasmic Caechanism Fig. 6A.


Both MCU  knockdown and pretreatment with ruthenium red, an inhibitor of MCU, inhibited celastrol-induced mitochondrial Ca2+ uptake, dilation of mitochondria/ER, accumulation of poly-ubiquitinated proteins, and cell death in MDA-MB 435S cells. Inhibition of the IP3 receptor (IP3R) with 2-aminoethoxydiphenyl borate (2-APB) also effectively blocked celastrol-induced mitochondrial Ca2+ accumulation and subsequent paraptotic events. Collectively, our results show that the IP3R-mediated release of Ca2+ from the ER and its subsequent MCU-mediated influx into mitochondria critically contribute to celastrol-induced paraptosis in cancer cells.Celastrol, a triterpene extracted from the Chinese “Thunder of God Vine”, is known to have anticancer activity, but its underlying mechanism is not completely understood. In this study, we show that celastrol kills several breast and colon cancer cell lines by induction of paraptosis, a cell death mode characterized by extensive vacuolization that arises via dilation of the endoplasmic reticulum (ER) and mitochondria. Celastrol treatment markedly increased mitochondrial Ca Tripterygium wilfordii i), which peaked at 3 h post-treatment  showed that celastrol treatment also increased the mitochondrial Ca2+ levels ([Ca2+]m), which peaked at 2 h post-treatment  and the ryanodine receptor (RyR) [3R [2+]m  , showed RyR) i, particularly [Ca2+]m, in our tested cancer cells. While the physiological [Ca2+]m signal regulates [Ca2+]c and stimulates oxidative metabolism, excess mitochondrial Ca2+ accumulation causes cell stress to leading to cell death c, treated cells were incubated with 2.5 μM Fluo-3-AM at 37°C for 20 min, washed with HBSS (without Ca2+ or Mg2+), and analyzed immediately by flow cytometry. To measure [Ca2+]m, treated cells were incubated with 2.5 μM Rhod-2-AM at 4°C for 30 min, washed with HBSS (without Ca2+or Mg2+), further incubated with HBSS at 37°C for 20 min, and then analyzed by flow cytometry and fluorescence microscopy. To confirm the mitochondrial localization of the Rhod-2 probe, cells were loaded with 2.5 μM Rhod-2-AM in HBSS (without Ca2+ or Mg2+) for 30 min at 4°C. The cells were then washed with HBSS and visualized by the fluorescence microscopy using Zeiss filter sets #46 and #20.To measure [CaCells were prefixed in Karnovsky's solution  for 2 h and washed with cacodylate buffer. Post-fixing was carried out in 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 h. After dehydration with 50-100% alcohol, the cells were embedded in Poly/Bed 812 resin (Pelco), polymerized, and observed under electron microscope .t test was applied to evaluate the differences between treated and control groups with cell viability. Data from multiple groups were analyzed by one-way ANOVA, followed by Bonferroni multiple comparison test. For all the tests, the level of significance was values of P < 0.05.All data were presented as mean ± S.D. (standard deviation) from at least three separate experiments. Student's


Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) . In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically.Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Ca Baseline readings were obtained on exposure to HEPES-buffered saline, and Ca2+ homeostasis was monitored in response to the addition of a drug, or in response to Ca2+-free HEPES-buffered saline .WT HEK293 or HEK293/Cat tests, one-way ANOVA with a multiple comparison test or repeated measures one-way ANOVA with a multiple comparison test.Statistical comparisons were made using, as appropriate, paired or unpaired student’s 2+ channels in proliferation . To study L-type Ca2+ currents, we used a holding potential of −50 mV (in order to inactivate T-type Ca2+ channels) and replaced Ca2+ with Ba2+ to promote influx via L-type rather than T-type Ca2+ channels. Under these conditions, currents displaying little or no inactivation were also inhibited by CORM-2 but not iCORM . Thus, CO can inhibit both T-type and L-type Ca2+ channels natively expressed in A7r5 cells.Exposure of A7r5 cells to CoPPIX caused a concentration-dependent increase in the expression of HO-1, as detected by Western blotting Fig. . This prORM Fig. . Where tORM Fig. , these cORM Fig.  and, wheORM Fig. , were inTo examine whether the HO-1/CO pathway was able to modify proliferation in human VSMCs, we studied cells cultured from human saphenous vein. Figure 2+ channel blocker, mibefradil  significantly reduced proliferation  caused a fall of [Ca2+]i which was far larger than that seen in WT cells , which caused a striking and largely irreversible reduction of [Ca2+]i in Cav3.2-expressing HEK293 cells, but not in WT cells  been based on the use of mibefradil  Ca2+ channel knock-down i  at or around the cell’s resting membrane potential. The presence of a window current generated by expressed Cav3.2 channels would also require that native currents set the resting membrane potential appropriately. The nature of such currents has not to date been explored in depth.T-type Ca cancers , where t cancers ). The pr cancers , that ov+]i Fig. , suggestcurrent’  which ar−/− mice [−/− mice prevented hypoxic pulmonary remodelling and inhibited hypoxic VSMC proliferation in vitro, whereas biliverdin was ineffective [Numerous studies have indicated that HO-1 induction affords protection in a variety of pathological systemic cardiovascular conditions, many of which involve VSMC proliferation , 44. Ind−/− mice , and tar−/− mice . Clearly−/− mice . More reffective .2+ channels. Since CO can regulate all three isoforms of T-type Ca2+ channels , this r


Chronic lymphocytic leukemia is the most common leukemia in the United States. It is a slowly progressive disease, with an 82% five-year survival rate. The treatment strategies are highly individualized with patients in the early and stable stages typically not requiring treatment. However, those with progressive or clinically advanced disease will require treatment. Cytotoxic drugs, such as the alkylating agents, purine nucleoside antagonists, and immunotherapeutic agents, have been the mainstay of chemotherapeutic treatment in CLL. However, given the lack of therapeutic specificity, these medications  have limited tolerability due to side effects. In this paper, we will discuss the data on the use of phosphatidylinositol 3 kinase inhibitor Idelalisib in the management of patients with chronic lymphocytic leukemia. The preclinical and clinical data thus far demonstrate that Idelalisib produces a dramatic and durable response in patients with chronic lymphocytic leukemia and without causing significant toxicity. Moving forward, the ongoing clinical trials will help address the various questions currently being raised regarding the long-term application and safety of Idelalisib. With greater clinical experience following more widespread use of Idelalisib, we will be able to determine the optimal combination therapies in treatment-naïve and relapsed/refractory patients, resulting in more individualized therapeutic strategies for patients with chronic lymphocytic leukemia. Chronic lymphocytic leukemia (CLL) is a lymphoid malignancy characterized by the accumulation and proliferation of nonfunctional and monoclonal small CD5/CD19/CD-20/CD23-positive lymphocytes in the blood, bone marrow, and lymphoid tissues . DiagnosCLL is a slowly progressive disease, with an 82% five-year survival rate . The treUnfortunately, however, despite the availability of various therapeutic agents for CLL, the disease is currently considered incurable with most patients eventually relapsing after initial treatment. The poor outcomes of the current treatment strategies, especially in patients with high-risk features , and the lack of tolerability of cytotoxic drugs by the older patients have prompted research into the development of novel drug therapies , 7. The δ) inhibitor Idelalisib , the clinical rationale behind its use in the treatment of CLL, and the outcomes of various preclinical and clinical trials studying the use of Idelalisib alone or in combination with other therapeutic agents for treatment of both initial and relapse/refractory cases of CLL.In this review, we particularly focus on the phosphatidylinositol 3 kinase /Ig β  chains [κB pathway, and ERK pathway, leading to B-cell maturation and differentiation [The BCR is transmembrane receptor protein composed of two parts: an antigen-specific membrane bound immunoglobulin (Ig) and an intracellular signal transduction moiety—Ig α Igα, CD9A/Ig β  chains . BCR act) chains –14. BCR ntiation , 14.α, β, γ, and δ, act by phosphorylation of several membrane inositol phospholipids. Each of the class I PI3K isoforms is heterodimer molecules comprising a catalytic subunit p110  and a regulatory subunit p85  [PI3K are lipid kinases that play a central role in normal cellular functions such as cell growth, differentiation, proliferation, and survival . Constitnd p101) , 17.γ (PLCγ2) are recruited [γ2 catalyzes the hydrolysis of PIP2  to release the second messengers DAG  and PIP3 . PIP3 is responsible for the reorganization of the cell cytoskeleton and regulates cellular functions such as cell adhesion via calcium flux into the cells, whereas DAG activates several isoforms of protein kinase C and other downstream signal mediators [δ PI3K signaling pathway can also be activated by receptors other than the BCR such as by cytokines IL-4 [Binding of an antigen to the antigen-specific extracellular component of the BCR leads to phosphorylation of ITAMs (immunoreceptor tyrosine-based activation motifs) by tyrosine kinases LYN and spleen tyrosine kinase (SYK). ITAMs are intracytoplasmic specific sequences of the CD79A/B part of BCR which are responsible for further downstream signal cascades in B cells . Upon anecruited . PLCγ2 cediators . The p11nes IL-4 , IL-6, anes IL-4 , 21, by nes IL-4 , and by nes IL-4 –26.δ (p110δ) is mainly expressed in circulating leukocytes and lymphoid tissues along with the ubiquitously expressed PI3Kα [δ knock-out (KO) and p110δ knock-in (KI) mice that p110δ is essential for B-cell survival, migration, and proliferation. Mice deficient in a functional p110δ protein produced fewer mature B cells and exhibited impaired function in B cells that do manage to develop [δ is further highlighted by the finding that complete deficiency of PI3Kδ in cells makes them prone to apoptosis [δ isoform is introduced into cells lacking BCR expression, they are rescued from apoptosis [δ isoform as a wild-type protein induces transformation and constitutive activation of the AKT signaling pathway in cultured cells [PI3Ked PI3Kα . It has  develop . Additio develop . The imppoptosis , whereaspoptosis . Furthered cells .The above findings are a clear indication of the importance that the PI3K pathway has not only in normal B-cell development but for the proliferation, survival, and migration of CLL cells.δ in contrast with normal B-cells [δ, provides prosurvival signals to CLL cells and also leads to their homing and retention in the bone marrow and lymphoid tissue. Idelalisib (GS-1101 or CAL 101) is a PI3Kδ isoform specific kinase inhibitor. It has been reported that in vitro Idelalisib, via inactivation of PI3Kδ, leads to a decrease in AKT and ERK phosphorylation thus interrupting downstream BCR signaling. It induces apoptosis in CLL cells in a time- and dose-dependent manner via a caspase dependent pathway [In CLL tumor cells, there is increased activity of PI3K B-cells , 18. The pathway , 28. Thi pathway .γ by these immune effector cells resulting in impaired CLL cell proliferation [α, CD40L, BAFF, and fibronectin, all of which act by increasing AKT phosphorylation in CLL cells and protecting them from spontaneous apoptosis [Data from preclinical and clinical studies has shown that Idelalisib shows selective cytotoxicity to CLL cells compared with normal B cells. Furthermore, unlike pan-PI3K inhibitors, Idelalisib is not significantly cytotoxic to normal NK and T cells . Howeverferation , 29. Morpoptosis , 29.α, and fibronectin. This provides a strong rationale for the use of Idelalisib as a therapeutic agent in CLL patients.These findings demonstrate that Idelalisib targets multiple mechanisms of CLL cell survival by not only direct inhibition of the PI3K signaling pathway but also by inhibition of extrinsic activation of this pathway by microenvironmental stimuli from CD40L, BAFF, TNF-Multiple preclinical and clinical studies are being performed to investigate Idelalisib as a therapeutic agent for CLL as monotherapy or in combination with Rituximab and/or Bendamustine, Fludarabine, Ofatumumab, Chlorambucil, Chlorambucil + Rituximab, and Lenalidomide + Rituximab. A study investigating the synergistic effect of Idelalisib in combination with histone deacetylase inhibitor (HDI), panobinostat (LBH589), and suberoylanilide hydroxamic acid (SAHA) in vitro has also been performed in CLL and primary non-Hodgkin lymphoma (NHL) . The stuThe first phase I trial on Idelalisib studied the clinical pharmacokinetics of the drug in healthy volunteers and in those with lymphoid malignancies . FollowiN = 55), indolent non-Hodgkin lymphoma (N = 63), mantle cell lymphoma (N = 40), diffuse large B-cell lymphoma (N = 9) as well as in acute myelogenous leukemia (N = 12), and multiple myeloma (N = 12) has shown that responses were obtained in patients with CLL (4/17), indolent lymphoma (9/15), and mantle cell lymphoma (6/7) at all dose levels [Preliminary reports from a phase I trial studying the clinical activity of Idelalisib in relapsed or refractory B-cell malignancies such as chronic lymphocytic leukemia  is seen with Idelalisib therapy. According to the IWCLL response criteria , lymphoc2 weekly for 8 doses and/or 90 mg/m2 Bendamustine on days 1 and 2 of each cycle for 6 cycles, overall response rates (ORR) for the Idelalisib + Rituximab, Idelalisib + Bendamustine, and Idelalisib + Rituximab + Bendamustine regimens were 78%, 82%, and 87%, respectively, and the one-year progression-free survival (PFS) rates were 74%, 88%, and 87%, respectively [In a recent study investigating combinations of Idelalisib with Rituximab and/or Bendamustine, Coutre et al. report that when Idelalisib (150 mg BID continuously) was given with Rituximab at a dose of 375 mg/mectively . Moreoveectively . Vos et ectively . The resectively .2 weekly × 8) or a total of 12 infusions of Ofatumumab . Those patients who were on treatment after 48 weeks were eligible to continue Idelalisib on an extension study. The ORR was 83% with 3 CRs (8%) and a median time to response of 1.9 months. The PFS for all patients and duration of response (DOR) were 20 and 19 months, respectively. Among the 11 patients with del(17p) and/or TP53 mutations, the response rate was 73% and the median PFS and DOR were 19 months. This study demonstrates that the combination of Idelalisib with anti-CD20 antibodies such as Rituximab or Ofatumumab has good clinical activity and tumor control along with an acceptable safety profile in patients with relapsed/refractory CLL. Phase III trials evaluating the efficacy and safety of Idelalisib in combination with these two anti-CD20 antibodies, Rituximab or Ofatumumab, are currently underway  [A phase I study evaluating Idelalisib in combination with Rituximab or Ofatumumab was conducted in patients with relapsed/refractory CLL . They we1659021) .2 weekly × 8 doses and Idelalisib 150 mg BID continuously for 48 weeks; those who completed 48 weeks without progression were enrolled in an extension study and continued to receive Idelalisib. The ORR was 96%, and the median time to response was 1.9 months with no on-study relapses. At 24 months, the estimated PFS was 91%. Most notably, all of the six patients with 17p deletion (del(17p)) responded with 1 CR and 5 PR. The AEs reported were diarrhea, pyrexia, chills, fatigue, rash, pneumonia, and elevated ALT/AST.In a phase II study, O'Brien at al. investigated the combination of Idelalisib with Rituximab in treatment-naive patients ≥65 years of age, with CLL or SLL . They weThere are several genetic mutations that serve as markers of poor prognosis in CLL including del(17p)/TP53 mutation, del(11q), IGHV mutation, and NOTCH1 mutation –46. CoutA combination of Idelalisib with CX-4945, an orally bioavailable selective inhibitor of Casein Kinase 2 (CK2), has also been tested and has shown promising activity in CLL . AnotherFurman et al. enrolled 220 patients with relapsed CLL and coexistent medical conditions such as decreased renal function, chemotherapy related bone marrow suppression, and other major comorbidities . The patTo date, the results from trials evaluating the clinical efficacy and safety of Idelalisib, both as monotherapy and as part of combination regimens, have been encouraging. However, there are still questions to be answered about the long-term application of Idelalisib, particularly about its safety, tolerability, drug interactions, and most importantly the duration and durability of response. There is a concern that, with specific inhibition of such a complex pathway as the PI3K pathway, there exists a potential for escape by upregulation or activation of other PI3K isoforms, resulting in drug resistance . Future The extraordinarily promising results shown by targeted therapy have revolutionized the current treatment strategies for CLL. The preclinical and clinical data thus far demonstrate that Idelalisib produces a dramatic and durable response in CLL patients without causing significant toxicity, which is a drawback of other nonspecific therapies. Moving forward, the ongoing clinical trials will help address the various questions currently being raised regarding the long-term application and safety of Idelalisib. With greater clinical experience following more widespread use of Idelalisib, we will be able to determine the optimal combination therapies in treatment-naïve and relapsed/refractory patients, resulting in more individualized therapeutic strategies for CLL patients.


Background: The Ca2+-releasing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) acts via lysosomal two-pore channels (TPC2).Results:Tpcn2−/− cardiac myocytes showed reduced acute responses to β-adrenoreceptor stimulation and chronically reduced cardiac hypertrophy and arrhythmogenesis.Conclusion: Acute and chronic effects of cardiac β-adrenoreceptor stimulation depend on NAADP acting via TPC2 in lysosomes.Significance: NAADP/TPC2 signaling pathways offer new strategies for cardiac therapeutics. 2+-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca2+ release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca2+ responses in cardiac myocytes from Tpcn2−/− mice, unlike myocytes from wild-type (WT) mice. Ca2+/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca2+ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2−/− mice. Increases in amplitude of L-type Ca2+ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2−/− mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2−/− mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2−/− mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼25 nm). We propose that Ca2+-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.Ca This isoform was targeted because it has been shown to be specifically expressed in lysosomal and late endosomal membranes  physiology of Cam: NaCl 136, KCl 5.4, NaHCO3 12, Na+ pyruvate 1, NaH2PO4 1, MgCl2 1, EGTA 0.04, glucose 5; gassed with 95% O2, 5% CO2 to maintain a pH of 7.4), digestion was carried out with a Tyrode's solution (50 ml) of identical composition, but which lacked EGTA and contained 24–28 mg of collagenase type II (Worthington) and 0.1 mm CaCl2. Following digestion, the left atrium was separated and placed in high K+ storage medium . Single cells were isolated by gentle trituration and stored at 4 °C in high K+ storage medium until use. Ventricular tissue was dissected into several pieces and underwent mechanical agitation for a further 5 min in collagenase solution. Isolated myocytes were filtered through a 250-μm mesh, and collagenase was replaced with Dulbecco's modified Eagle's medium  warmed to 37 °C. Ventricular myocytes were stored in DMEM at room temperature and used within 9 h of isolation.Male guinea pigs (300–500 g) were killed in accordance with Schedule 1 of The Animals (Scientific Procedures) Act 1986 (HMSO). The heart was rapidly excised and washed in a modified Tyrode's solution containing EGTA and heparin to prevent clots from forming in the small coronary circulation. The heart was then mounted on a constant pressure Langendorff perfusion system for retrograde perfusion via the aorta. Following a 2-min initial perfusion with a modified Tyrode's solution  solution containing (mm) NaCl 125, NaHCO3 25, KCl 5.4, NaH2PO4 1.2, MgCl2 1, CaCl2 1.0, glucose 5.5, pH 7.4, together with heparin (10 units/ml) and streptokinase (100 units/ml). The cannula was then removed from the syringe and mounted onto a Langendorff perfusion system. Once mounted, the hearts were perfused with a solution (36 °C) containing (mm) NaCl 130, KCl 5.4, MgCl2 3.5, glucose 10, HEPES 5, NaH2PO4 0.4; pH 7.4, and gassed with 95% O2, 5% CO2. After 3 min, this isolation solution was replaced with a further 50 ml of a similar solution that also contained 0.3 mg/ml collagenase  and 0.1 mm CaCl2. Following enzymatic digestion (for a maximum time of 7 min), the heart was removed from the cannula. The ventricles were cut into pieces, and the myocytes were released during several gentle suspensions, then filtered through 250-μm mesh, and stored at room temperature in medium containing (in mm) NaCl 130, KCl 5.4, MgCl2 3.5, CaCl2 0.1glucose 10, HEPES 5, NaH2PO4 0.4, taurine 20; 0.1% bovine serum albumin; pH 7.4, and gassed with 95% O2, 5% CO2.Mice (13–18 weeks old) were killed in accordance with Schedule 1 of The Animals (Scientific Procedures) Act 1986 (HMSO). Hearts were excised, tied to a cannula via the aorta and perfused through a syringe containing a pre-gassed (with 95% Om) NaCl 125, NaHCO3 25, KCl 5.4, NaH2PO4 1.2, MgCl2 1, glucose 5.5, CaCl2 1.8 and oxygenated with 95% O2, 5% CO2 to maintain a pH of 7.4. The PSS used for mouse experiments was identical except that CaCl2 concentration was reduced to 1 mm. All electrophysiology was performed at 36 °C. Glass microelectrodes were manufactured from thin-walled, filamented borosilicate glass capillary tubing . Electrode resistances of 3.0 ± 1.5 megohms were used for whole-cell experiments. During whole-cell recordings, a whole-cell patch solution was used containing(in mm) K+ aspartate 110, KCl 10, NaCl 5, MgCl2 5.2, HEPES 5, K2ATP 5, pH 7.2, with KOH, to which Ca2+ indicator dye, caged NAADP, and inhibitors were added at the stated concentrations. Perforated patch was carried out using whole-cell patch solution plus amphotericin B at 250 μg/ml. Current clamp recordings were made using an AxoClamp 2A amplifier system (Axon Instruments). Cells were stimulated to fire action potentials using a 2-ms current pulse applied via the microelectrode at a rate of 1 Hz. Voltage clamp recordings were made using an AxoPatch 200B amplifier system (Axon Instruments). To stimulate the L-type Ca2+ current (ICaL), cells were maintained at a holding potential of −40 mV and stimulated by a 200-ms step depolarization to 0 mV at a rate of 0.2 Hz. ICaL amplitude was measured as maximum current minus current at the end of the voltage pulse.Cardiac myocytes were mounted in a perfusion bath placed at the stage of an inverted microscope and perfused with a physiological salt solution (PSS) containing (in mm) was added to the whole-cell patch solution. In mouse experiments, myocytes were incubated with Fluo-5F AM (20 μm) for 20 min. Ca2+ transients were stimulated at 1 Hz either by a patch electrode  or by carbon fiber electrodes placed at the side of the superfusion bath (mouse experiments).During whole-cell fluorescence experiments, Fluo-5F salt . Photomultiplier signals were directed through an amplifier, followed by a low pass 100 Hz electronic filter, digitized , and recorded with pClamp software at an acquisition rate of 10 kHz.Guinea pig and mouse ventricular myocytes were visualized using a Nikon Axiovert 200 inverted microscope with attached Nipkow spinning disk confocal unit . Excitation light was provided by a 488-nm diode laser (Cairn Research Ltd.) passed though the Nipkow unit and delivered to the sample through the objective. Emitted light passed back through the CSU-10 unit and was detected using an Andor iXON897 EM-CCD camera (Andor Technology) at a frame rate of 50 Hz. Images were recorded and analyzed using Andor iQ software (Andor Technology).Mouse ventricular myocytes were imaged with a confocal microscope system that consisted of a Leica TCS NT scanning head coupled to a Leica DMIRB inverted microscope with a 100× oil immersion objective lens  in line scan mode (2.6 ms per line). Excitation light (488 nm) was provided by an air-cooled 488-nm argon ion laser system (Uniphase Ltd.), and emitted light was collected at wavelengths above 515 nm using a long- pass filter. Images were recorded using Leica TCS NT software and analyzed using ImageJ software.F/F0 such that fluorescence data are presented relative to diastolic fluorescence. In the case of experiments in which NAADP was photoreleased in guinea pig ventricular myocytes, there was a small baseline change, and data are presented as F/F0i where F0i is equal to diastolic fluorescence before photorelease of NAADP.For analysis, background fluorescence was subtracted, and multiple transients were averaged to collect data at a given time point. Data are presented as Rabbit ventricular tissue was fixed in Karnovsky fixative , resin-eCells were stimulated to contract via a patch electrode (permeabilized configuration), and contractile properties were studied using the IonOptix system (IonOptix Corp.) to measure sarcomere length. Cells were visualized via a ×40 oil objective using an IonOptix MyoCam (IonOptix Corp.) that sampled images at a frequency of 240 Hz. Sinusoidal optical density traces arising from the alternating light and dark bands of the contractile machinery were then transformed into a signal of sarcomere length by application of a fast Fourier transform by the IonWizard sarcomere length acquisition software (IonOptix Corp.). Length measurements were calibrated using a stage micrometer with 2-μm graduations such that the number of pixels/μm recorded by the MyoCam could be entered into the software as a fixed value. Amplitude of sarcomere shortening was calculated by deduction of systolic from diastolic sarcomere length and expressed as a percentage of resting sarcomere length. Analysis was performed using IonWizard 5 software (IonOptix Corp.). All values represent an average of 10 contractions.m CaCl2  at a constant flow rate (3.5 ml/min). Hearts were allowed to beat spontaneously, and contractile force was measured by means of a hook placed through the ventricles near the apex, attached to a tension transducer. Resting tension was set between 0.5 and 1.0 g initially. Heart rate was calculated from the force signal as the reciprocal of the time interval between contractions. Inflow pressure was measured using a pressure transducer positioned close to the aorta so that changes in coronary artery resistance could be measured as pressure changes at a constant perfusion flow rate. Data were acquired and analyzed using Chart software , with a data acquisition sampling rate of 100 samples/s. The tension signal was low pass-filtered with a cutoff frequency of 50 Hz.Mice were killed in accordance with Schedule 1 of The Animals (Scientific Procedures) Act 1986 (HMSO). Hearts were excised, cannulated via the aorta under a microscope, and perfused for 30 s with PSS solution containing streptokinase (100 units/ml) via a syringe connected to the cannula. After initial perfusion, the syringe was removed and the cannula mounted on a Langendorff apparatus for retrograde perfusion with PSS containing 1.8 mNPE-caged NAADP, synthesized in-house by a method described previously , was incNAADP-AM was synthesized in-house by a method described previously . LoadingTpcn2−/− mice were subjected to PES. Monophasic action potentials were recorded from the left ventricular epicardial surface.To assess propensity to arrhythmias, Langendorff-perfused hearts from WT and m isoproterenol. Ventricular pacing first occurred with a progressively increasing pacing current (from pacing capture current to 35 mA). At each current amplitude, three bursts of 50 stimuli were delivered. The cycle length between each stimulus was held at 20 ms, and the bursts were separated by a 2-s pacing-free interval. Data were expressed as a cumulative percentage of pacing-free time during which ventricular arrhythmias were observed. A second set of experiments was then performed in which the cycle length was progressively reduced (from 90 to 10 ms), and the pacing current was held at 10 mA. These data were analyzed using the same method.Hearts from mice not previously exposed to chronic β-adrenergic stimulation were subjected to PES in the presence of 50 nHearts from mice treated for 2 weeks with isoproterenol were subjected to a similar protocol in the absence of further β-adrenergic stimulation. A pacing train of eight stimuli (S1) was delivered with a cycle length of 100 ms, with a single (S2) premature extra stimulus introduced at progressively shorter intervals until arrhythmia was induced or the ventricular refractory period was reached. For the burst pacing protocol, ventricular pacing was carried out with a train of 50 S1 at a cycle length of 20 ms. Pacing current amplitude was progressively increased from the threshold of ventricular capture until ventricular tachycardia or fibrillation was induced or a current of 35 mA was reached. Ventricular tachycardia was defined as six or more consecutive premature ventricular waveforms .Tpcn2−/− mice.Cardiac hypertrophy was induced by administration of isoproterenol  at 10 mg/kg/day for 14 days via osmotic mini-pumps (Alzet) implanted subcutaneously in 8–10-week-old WT and Mice were anesthetized with Avertin  via intraperitoneal injection. Transthoracic M-mode echocardiographic recordings were performed using an Acuson Sequoia C256 system (Siemens) following a protocol described previously . Measurein vivo electrocardiographic analysis on mice anesthetized with isoflurane (2.5%). RR interval, P wave duration, PR interval, QRS, JT, and QT durations were recorded.To monitor cardiac rhythms, we carried out t tests or one- or two-way analysis of variance (with repeated measures if appropriate) followed by either Tukey, Bonferroni, or Dunnett's post hoc test. Where a data set could not be deemed normally distributed, a Mann-Whitney test was used instead. A statistically significant difference was concluded when p was < 0.05. All data are expressed as mean values ± S.E.Statistical comparisons were made using paired or unpaired Student's Tpcn2−/− mice, as shown in the inset to Fig. 1C (compared with wild type (WT)). Effects of exogenous NAADP were investigated in cells from these Tpcn2−/− mice by superfusing myocytes with NAADP-AM, a membrane-permeant acetoxymethyl ester of NAADP  caused a significant increase in the amplitude of these Ca2+ transients  in myocytes from WT mice, but there was a striking failure of NAADP-AM to increase Ca2+ transient amplitude in ventricular myocytes from Tpcn2−/− mice . No difference was seen in Ca2+ transient amplitude between the two genotypes under control conditions . Therefore, the expression of TPC2 proteins is required for NAADP-mediated enhancement of Ca2+ transients in cardiac myocytes.We first demonstrated the absence of TPC2 expression at the mRNA level in cardiac tissue from of NAADP , 33, 41.D–F, shows effects of isoproterenol (3 nm) on contraction of myocytes electrically stimulated at 1 Hz. Increases in contraction amplitude caused by β-adrenoreceptor stimulation were greatly reduced in myocytes from Tpcn2−/− mice  as compared with the increases observed in WT myocytes . Action potential recordings made during the contraction study showed no difference between WT and Tpcn2−/− cells under control conditions but showed a smaller increase in the “late plateau” phase of Tpcn2−/− action potentials during exposure to ISO .We then tested for the involvement of TPC2 in the response to β-adrenoreceptor stimulation. +/Ca2+ exchanger activity, the reduced effect of isoproterenol on contraction in myocytes lacking TPC2 proteins was hypothesized to arise from reduced effects on Ca2+ transients. This was investigated directly using the Ca2+ probe Fluo-5F. G and H, shows representative Ca2+ transients in myocytes stimulated at 1 Hz. In line with the contraction data, the effects of isoproterenol (3 nm) on Ca2+ transients in myocytes from Tpcn2−/− mice were significantly reduced compared with WT .In view of the evidence presented above, and given that the late plateau phase of the action potential is largely dependent on Na2+ influx through L-type channels  showing that the β-adrenoreceptor signaling pathway per se remained intact, allowing PKA to phosphorylate L-type Ca2+ channels in the usual way.Previous work has demonstrated that NAADP has no effect on Cachannels , 33. We Tpcn2−/− mice were complemented by observations in whole hearts perfused by the Langendorff technique. In spontaneously beating hearts, isoproterenol (5 nm) increased the force of ventricular contraction by 92 ± 4% (n = 5) in age-matched WT controls compared with 63 ± 9% (n = 7) in hearts from Tpcn2−/− animals, a significant reduction in animals lacking TPC2 .The observations in myocytes from Tpcn2−/− myocytes shows a striking parallel to additional observations made during pharmacological suppression of the NAADP pathway in guinea pig ventricular myocytes; exposure to bafilomycin A . Although suppression of the NAADP pathway reduced the ability of isoproterenol to increase Ca2+ transients, the effect of isoproterenol on L-type Ca2+ currents was unaffected by Ned-19 showing that other stages of the β-adrenoreceptor pathway remained operational (E–G and I).The reduced response to isoproterenol in  loading , 33) or of NAADP ) both re2+ transients and contractions in ventricular myocytes from mouse and guinea pig.Taken together, the observations reported above provide a compelling case for the contention that NAADP, most likely acting via the TPC2 protein, plays a major role in the acute effects of isoproterenol on the magnitude of stimulated Ca2+ transients triggered by action potentials are associated with an increased Ca2+ load of the SR . Following photorelease of NAADP from a caged NPE derivative  similar to those described previously  . KN-92 is a structurally related analogue of KN-93 without effects on CaMKII, and this substance failed to suppress the effects of photoreleased NAADP in atrial myocytes (C and E).Previous work has shown that the effects of NAADP to increase the amplitude of Caf the SR , 33. Thia2+ load . This warivative , there w, and E) , 33. Themyocytes , C and E2+ transients were increased following exposure to NAADP-AM in the absence (n = 20) but not the presence of KN-93 . Similar observations were made in guinea pig ventricular myocytes ). These observations are therefore consistent with a role for CaMKII in the pathway-mediating effects of NAADP in guinea pig and mouse myocytes, in part through phospholamban/SERCA, although additional effects, for example on ryanodine receptors, cannot be excluded.Effects of the active KN-93 were also tested in ventricular myocytes from WT mice exposed to NAADP-AM. Again, Ca n = 10, , D–F. Si2+ uptake (A) and in locations thought to mediate Ca2+ release via RyRs (B). The mean distance of lysosomes from the SR was 25.4 ± 1.8 nm (n = 30). The significance of these observations is considered in more detail under the “Discussion.”For an amplification mechanism to work in the way we have hypothesized above, a close structural relationship would be required to exist between lysosomes and the SR. This was investigated using transmission electron microscopy of rabbit ventricular tissue, and sample images are shown in + uptake A and in via RyRs B. The me2+ transients in electrically stimulated myocytes, and taking into account the finding that arrhythmias caused by isoproterenol could be suppressed by the NAADP antagonist BZ194 (Tpcn2−/− mice were significantly (p < 0.001) less prone to arrhythmias when subjected to a ventricular burst pacing protocol compared with WT controls. The induction threshold for ventricular tachycardia or fibrillation using a burst pacing protocol in hearts exposed to isoproterenol was significantly higher in hearts from Tpcn2−/− mice compared with hearts from WT mice . In particular, the increase in heart weight after chronic exposure to isoproterenol (expressed either as left ventricular mass or the ratio of heart weight to tibia length) was significantly less in Tpcn2−/− mice than in WT . Additional ex vivo electrophysiological studies indicated that hearts from isoproterenol-treated Tpcn2−/− mice showed less susceptibility to arrhythmia when subjected to a ventricular burst pacing protocol in the absence of further acute isoproterenol exposure .Building upon the above observations showing a role for NAADP and TPC2 proteins in the acute effects of β-adrenoreceptor stimulation and acute arrhythmogenesis, we investigated whether NAADP-mediated Caistology , A–C. In2+ signaling require the presence of functional CaMKII. TPC2 proteins also appear to contribute to arrhythmogenic effects during excessive acute β-adrenergic stimulation. In addition, our work highlights the contribution of this pathway during chronic β-adrenergic stimulation leading to pathological hypertrophy and suggests that its inhibition is partially protective against the negative consequences of chronic activation of β-adrenergic signaling pathways.The observations described in this study demonstrate the requirement for TPC2 proteins in order for NAADP actions in mammalian cardiac tissue, and they highlight the contribution of these proteins during acute and chronic actions of the β-adrenergic signaling pathway. We have shown by using both genetic and pharmacological techniques that the NAADP pathway is physiologically important during acute β-adrenergic stimulation. Furthermore, pharmacological observations show that the actions of NAADP on cellular Ca2+ signals is an emerging field. Ca2+ release from lysosomes dependent on TPC2 has been demonstrated to regulate a disparate number of cellular processes, and it is the principal way in which NAADP evokes intracellular Ca2+ signals. Roles for TPCs in pathophysiological processes to date include triggering of the acrosome reaction in mammalian fertilization  and also the mitochondrial membrane and the SR . These relationships are not simply a structural coincidence; they not only facilitate major physiological processes, such as excitation-contraction coupling and regulation of oxidative phosphorylation respectively, but recent work has suggested molecular tethering between specific proteins on these structures to maintain their close apposition .Our results suggest that these lysosome-SR microdomains also exist in cardiac tissue. In the future, it will be important to investigate whether there is a preferential location of lysosomes that bear TPC2 proteins in the heart, as this would indicate where microdomains involved in NAADP-related CaThe proposed novel effects of CaMKII as part of the cardiac NAADP signaling pathway provide an additional mechanism for the extensive contributions of CaMKII to β-adrenoreceptor signaling in the heart . A role 54–2+ channels (to promote Ca2+ entry) and phospholamban (speeding relaxation and increasing the amount of Ca2+ stored in the SR). To this scheme, we would add that NAADP synthesis is also increased during β-adrenergic stimulation (2+ currents, RyR, and phospholamban. PKA-mediated phosphorylation of phospholamban is a major mechanism underlying the increased Ca2+ load of the SR following β-adrenoreceptor stimulation (2+ transients and contraction are also associated with an increase in SR Ca2+ load in both atrial and ventricular myocytes (Our observations can be readily incorporated in the accepted scheme that β-adrenoreceptor agonists lead to stimulation of adenylyl cyclase, formation of cAMP, and activation of PKA, followed by phosphorylation of proteins, including L-type Camulation , 32. A fmulation , 60. Givmulation , 61, 62 mulation , 63, altmulation , 53, 54.myocytes , 33.The mechanisms underlying cardiac hypertrophy following chronic β-adrenoreceptor stimulation are likely to be complex, but the evidence reported here supports a role for TPC2 proteins.In summary, our observations provide strong support for a role for NAADP and TPC2 proteins as major contributors in the actions of β-adrenoreceptor agonists to increase contraction amplitude in cardiac ventricular myocytes and suggest that these effects require the presence of functional CaMKII. TPC2 proteins also appear to play a major role in the pathway linking the stress of chronic stimulation of β-adrenoceptors to hypertrophy and associated tendency to arrhythmias. These proteins are likely to provide a promising new target for future therapy, because inhibiting the NAADP/TPC2 pathway has minimal effect on normal excitation-contraction coupling but suppresses adrenergic signaling in the heart.Tpcn2−/− physiology; cell isolations were carried out by E. L. B., R. A. C., and W. K. L. W. K. L. synthesized NAADP-AM; D. B-Y. and G. C. C. synthesized caged NAADP. M. R., J. P., and A. G. were responsible for the development of the Tpcn2−/− mouse line and subsequent breeding. K. T. S. carried out confirmation of Tpcn2−/− mRNA. R. A. B. B. and D. A. performed electron microscopy studies. A. G., M. R., and D. A. T. were responsible for experimental design, with assistance from R. A. C., E. L. B., W. K. L., and X. W. W. L. carried out echocardiography. D. A. carried out experiments on arrhythmias during acute isoproterenol stimulation. Y. W. carried out osmotic minipump experiments and chronic isoproterenol stimulation work. All experiments were carried out in the laboratories of D. A. T. and M. R. All authors contributed to the writing and editing of the manuscript.R. A. C. carried out all guinea pig work; E. L. B. and W. K. L. carried out all


Insulin promotes glucose uptake by cardiomyocytes and its use as a substrate for glycolysis and mitochondrial oxidation in order to maintain the high cardiac energy demands. Insulin stimulates Ca2+ signaling in normal and norepinephrine or insulin like growth factor–1-induced hypertrophic cardiomyocytes. Using mitochondrion-selective Ca2+-fluorescent probes we showed that insulin increases mitochondrial Ca2+ levels. This signal was inhibited by the pharmacological blockade of either the inositol 1,4,5-triphosphate receptor or the mitochondrial Ca2+ uniporter, as well as by siRNA-dependent mitochondrial Ca2+ uniporter knockdown. Norepinephrine-stimulated cardiomyocytes showed a significant decrease in endoplasmic reticulum-mitochondrial contacts compared to either control or insulin like growth factor–1-stimulated cells. This resulted in a reduction in mitochondrial Ca2+ uptake, Akt activation, glucose uptake and oxygen consumption in response to insulin. Blocking mitochondrial Ca2+ uptake was sufficient to mimic the effect of norepinephrine-induced cardiomyocyte hypertrophy on insulin signaling.In the present study we investigated insulin-dependent mitochondrial Ca2+ uptake is a key event in insulin signaling and metabolism in cardiomyocytes.Mitochondrial CaThe online version of this article (doi:10.1186/s12964-014-0068-4) contains supplementary material, which is available to authorized users. Cardiac hypertrophy is a physiological process that occurs in response to an increase in heart workload. Initially, it can be compensatory, however, a chronic elevated workload, the neurohumoral input, or an underlying genetic mutation can lead to pathological hypertrophy, contractile dysfunction, ventricular dilatation, and finally, heart failure . Moreove2+ is known to play a key role  evoked with both stimuli, CCCP and Thapsi. Moreover, to further confirm the involvement of MCU in our model we used a complementary approach to alter MCU activity that was independent of any nonspecific effects of Ru. Cardiomyocytes were transiently transfected with a small interfering RNA (siRNA) against MCU, reaching a knockdown of 72%, 72 h post transfection  for 24 h to induce hypertrophy  compared with control conditions. Importantly, this inhibitory effect appears not to be operative in IGF-1-treated cardiomyocytes  and thus is not simply a requisite change in response to cell growth. Moreover, histamine-dependent mitochondrial Ca2+ uptake was decreased in NE-treated cardiomyocytes, suggesting that changes in mitochondrial Ca2+ uptake in NE-treated cardiomyocytes occur downstream of Ca2+ release from ER and are not due to an upstream change in the insulin signaling. Interestingly, Ca+2 release from the ER after Thapsi treatment is augmented in NE-treated cardiomyocytes in comparison to control and IGF-1-treated cardiomyocytes, further indicating that the reduction in mitochondrial Ca2+ uptake is not due to a reduction in ER Ca2+ loading , as illustrated in the upper left hand box of Figure 3R as the primary Ca2+ channel involved in Ca2+ transfer to the mitochondria, we evaluated the subcellular distribution of the most expressed InsP3R type 2 isoform (InsP3R2) and the mitochondrial marker mtHsp70. Figure 3R2 and the mitochondrial marker mtHsp70 in NE-treated cardiomyocytes compared to vehicle treated controls. In this case, both M1 and M2 were significantly reduced. We realize that there are important differences between the ER-mitochondrial interface in a functioning heart compared to cultured cells. Therefore a comprehensive validation of the differences between the two conditions is beyond the scope of our current manuscript, and although we believe that our findings are relevant to in vivo events, clearly further experimentation will be required to validate this.The distance between ER and mitochondria is a key determinant of Caganelles . To evalganelles ,31 which2+ uptake and mitochondrial metabolism seen under certain pathological hypertrophic states.Next, we tested if the reduction in ER-mitochondria coupling in NE-treated cardiomyocytes correlated with a change in mitochondrial metabolism. The rate of oxygen consumption was significantly reduced in NE-stimulated cardiomyocytes compared to controls.  Figure H. Oxygenin vitro cell culture model of NE-induced hypertrophy, various cellular processes regulated by insulin were measured. Akt is a serine/threonine kinase downstream of insulin receptor whose activity is regulated by phosphorylation 2-deoxyglucose [Cardiomyocytes were rinsed twice with HEPES-buffered saline. Inhibitors were added for 30 min and insulin during the last 10 min. Glucose uptake was measured using 10 mM [yglucose .ΔΨm was measured after loading cardiomyocytes with TMRM (200 nM) for 30 min. Afterward, cells underwent trypsinization, and fluorescence was assessed by flow cytometry (excitation/emission 543/560) with a FACScan system (Becton Dickinson). CCCP (50 mM) and oligomycin (10 mM) for 30 min were used as positive and negative controls for the ΔΨm measurements.Real-time PCR was performed with SYBR green (Applied Biosystems) as previously described . Data fot test. A value of P < 0.05 was chosen as the limit of statistical significance.Data are presented either as means ± SEM of a number (n) of independent experiments or as examples of representative experiments performed on at least three separate occasions. Data were analysed by analysis of variance and comparisons between groups were performed using a protected Tukey


Different oncogenes and deregulation of tumor suppressors exploit this mechanism to favor the survival of altered, malignant cells. Two recent studies of the Pinton team revealed a novel, non-transcriptional function of cytosolic p53 in cell death. During cell stress, p53 is recruited to the ER and the ER-mitochondrial contact sites. This results in augmented ER Ca2+ levels by enhancing sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) activity, ultimately promoting mitochondrial Ca2+ overload. The boosting of “toxic” Ca2+ signaling by p53 appears to be a critical component of the cell death-inducing properties of chemotherapeutic agents and anti-cancer treatments, like photodynamic stress. Strikingly, the resistance of p53-deficient cancer cells to these treatments could be overcome by facilitating Ca2+ transfer between the ER and the mitochondria via overexpression of SERCA or of the mitochondrial Ca2+ uniporter (MCU). Importantly, these concepts have also been supported by in vivo Ca2+ measurements in tumor masses in mice. Collectively, these studies link for the first time the major tumor suppressor, p53, to Ca2+ signaling in dictating cell-death outcomes and by the success of anti-cancer treatments.Ca Buffering intracellular Ca2+ prevented this in vivo photodynamic stress-induced tumor cell death. These events critically depended on p53, since p53-deficient tumor cells failed to display photodynamic stress-induced [Ca2+] rise and cell death.Yet, until now, the role of Ca cancers , remaine-workers , 46 addrres Fig. . First, in vivo experiments underpin the central role of ER-mitochondrial Ca2+ transfers not only in cell death but also in therapeutic responses to anti-cancer strategies. In addition, they underscore the potential of potentiating Ca2+-transport systems at the level of the ER and the mitochondria to overcome cell-death resistance of tumors (like p53-deficient cancers) to therapeutic treatments. These studies also highlight the importance of considering and investing in the future application of Ca2+-signaling-based therapies to increase the therapeutic success of anti-cancer treatments. In particular, treatments based on inducing tumor cell death by phototherapy and likely also chemotherapy, could be boosted by activation of toxic Ca2+-signaling. The search for molecules that could improve ER-mitochondrial Ca2+ fluxes, being it activators of SERCA, IP Rs, VDAC1 or MCU, may turn out to be promising tools in patients that suffer from tumors that poorly respond to therapeutic regimens. Given the central role of Ca2+ signaling in a variety of physiological processes, an important challenge will be to avoid toxic effects in unaltered, healthy cells. Strategies could involve the local release of compounds using coated nanoparticles or their coupling via a protease-sensitive linker sequence to a chemical moiety. This would allow the compound to enter cancer, but not healthy, cells expressing the particular protease. This strategy has been successfully used for analogues of thapsigargin, a high-affinity, irreversible inhibitor of SERCA [2+-signaling” context of cancer cells due to deranged expression of Ca2+ channels and pumps or deranged signaling pathways involved in the activation of these channels, like arachidonic acid signaling towards plasmalemmal Orai1/Orai3 channels in prostate cancers [3 signaling towards type 2 IP3Rs in B-cell malignancies [2+-signaling drugs” that could enhance Ca2+ fluxes in (a subset of) cancer, but not in healthy, cells, thereby killing or sensitizing these cancers, including resistant p53-deficient cancers, to treatments.These elegant of SERCA . Further cancers  and IP3 gnancies . Indeed,gnancies , 50. Thignancies . These i2+ signaling from the ER into the mitochondria, critically depends on the presence of p53. These data reveal that pro-apoptotic Ca2+ signaling is an important factor that determines the success of these strategies to kill cancer cells, including those related to oncogenic p53 mutants. This work also provides good hope that promoting Ca2+ signaling from the ER into the mitochondria could be beneficial for the targeting of a variety of tumors, including tumors resistant to chemotherapy, and re-establishing sensitivity of malignant cells to anti-cancer therapies.In summary, these recent findings by Pinton and co-workers elucidate that the ability of chemo-/photo-therapies to trigger pro-apoptotic Ca