In the present study, we found that treatment of CGNs with GDF-15 for 24 h increased the intracellular Ca2+ concentration ([Ca2+]i) in response to membrane depolarization, as determined by Ca2+ imaging. Whole-cell current recordings indicated that GDF-15 increased the inward Ca2+ current (ICa) without altering steady-state activation of Ca2+ channels. Treatment with nifedipine, an inhibitor of L-type Ca2+ channels, abrogated GDF-15-induced increases in [Ca2+]i and ICa. The GDF-15-induced increase in ICa was mediated via up-regulation of the Cav1.3 α subunit, which was attenuated by inhibiting Akt/mTOR and ERK  pathways and by pharmacological inhibition of Src-mediated TβRII phosphorylation. Given that Cav1.3 is not only a channel for Ca2+ influx, but also a transcriptional regulator, our data confirm that GDF-15 induces protein expression via TβRII and activation of a non-Smad pathway, and provide novel insight into the mechanism of GDF-15 function in neurons.GDF-15 (growth/differentiation factor 15) is a novel member of the TGF (transforming growth factor)-β superfamily that has critical roles in the central and peripheral nervous systems. We reported previously that GDF-15 increased delayed rectifier outward K GDF-15 (growth/differentiation factor 15), also known as macrophage inhibitory cytokine-1, is a distant member of the TGF (transforming growth factor)-β superfamily . Recent in vivo, promoting the survival of damaged mesencephalic dopaminergic neurons following cortical lesioning i activate signalling pathways that induce the expression of genes essential for dendritic development, neuronal survival and synaptic plasticity [2+]i also regulates gene expression during CGN development [2+]i in CGNs and the mechanisms that are involved is unknown. In the present study, we evaluated the effect of GDF-15 on [Ca2+]i using Ca2+ imaging while simultaneously recording inward Ca2+ current (ICa), since changes in [Ca2+]i in CGNs are associated with Ca2+ influx-dependent Ca2+ release i in single cells was detected on the basis of fura 2 fluorescence intensity as described previously i was determined for 60 s immediately before the addition of high-K+ solution (27 mM KCl). Fluorescence intensity was quantified using Metafluor software .i by Ca2+ imaging using the Ca2+-sensitive fluorescent dye fura 2. Since GDF-15 did not affect basal [Ca2+]i, we used a high-K+ solution (27 mM KCl) to depolarize neurons and activate VGCCs (voltage-gated Ca2+ channels), inducing a rapid increase in [Ca2+]i. In control neurons, depolarization with high K+ caused acute elevation of [Ca2+]i, with an increase in the F340/F380 ratio from 0.69±0.01 (n=43) to a maximum of 1.71±0.06 (n=127). After treatment with GDF-15 for 24 h, the ratio increased to a maximum value of 2.24±0.01 (n=115) (We demonstrated previously that GDF-15 increases  (n=115) A and 1B, (n=115) C.ICa, which was evoked by a 200-ms depolarization from a holding potential of −80 to 10 mV. GDF-15 application (100 ng/ml for 24 h) increased the ICa amplitude by 44.57%   curves of control and GDF-15-treated CGNs showed that ICa increased from a negative potential of −40 mV to a maximum value of 10 mV   A and 2B.ntervals C. The cuof 10 mV D, indica > 0.05) E. These I–V curves suggested that the Ca2+ channels were L-type channels found in neurons i evoked by high K+ and inhibited the GDF-15-induced increase in [Ca2+]i (F340/F380 ratio evoked by high-K+ solution without and with GDF-15 was reduced from 1.71±0.06 (n=127) to 1.15±0.10 (n=58) and from 2.24±0.01 (n=115) to 1.23±0.07 (n=45) respectively (ICa evoked by a 200-ms depolarization from −80 to 10 mV by 29.95±5.9% (n=24 and 7), and abolished the GDF-15-induced increase in ICa amplitude (2+ channels mediate the GDF-15-induced increase in the ICa amplitude and [Ca2+]i.The  neurons . To dete (10 μM)  abrogate [Ca2+]i A and 3B.ectively C. Consismplitude A and 4B,ICa is due to an up-regulation of channel expression. A previous study showed that Cav1.2 and Cav1.3 are the major α-subunits of L-type Ca2+ channels i, which involved activation of the same receptor and some of the same downstream signalling components as those previously reported by our group.GDF-15 plays various roles in neuroprotection, neural regeneration and axonal elongation ,8,9. How2+ signals. In neurons, VGCCs are L-, N-, P/Q-, R- and T-type Ca2+ channels i in response to membrane depolarization and nifedipine-sensitive ICa were up-regulated, suggesting the involvement of L-type Ca2+ channels, although we cannot exclude the possibility that nifedipine-insensitive Ca2+ channels or N-, P/Q- or R-type Ca2+ channels were also modulated by GDF-15. A study of rat CGNs indicated that administration of PACAP (pituitary adenylate cyclase-activating polypeptide) induced a rapid rise in [Ca2+]i and thereby stimulated Ca2+ influx through N-type but not L-type Ca2+ channels [2+]i, but not the response to membrane depolarization. Moreover, PACAP modulated the channels through rapid phosphorylation of channel proteins rather than regulation of Ca2+ channel α subunit expression [VGCCs are voltage sensors that convert membrane depolarization into intracellular Cachannels ,24. L-ty in CGNs ,29. Ca2+eostasis ,30. Our channels . This dipression .v1.1–Cav1.4. Cav1.1 and Cav1.4 are mainly expressed in skeletal muscle and retinal cells [v1.2 and Cav1.3 are abundant in the brain [v1.2 and Cav1.3 account for 89% and 11% of L-type channel transcripts respectively in mouse CGNs, and Cav1.2 comprises the pore-forming subunits of anomalous L-type channels in these cells [2+ channels coexist in rat CGNs [v1.3 α subunit than what is predicted from mRNA levels, leading to a higher number of functional Cav1.3 channels in the membrane.L-type channels consist of subtypes Caal cells ,33, wherhe brain ,34. Bothhe brain ,36, wherhe brain –39. Cav1se cells . Howeverrat CGNs . Besidesv1.3 and Cav1.2 differ in terms of biophysical properties, distribution in the brain and function [v1.3 protein but not Cav1.2 protein, but both Cav1.2 and Cav1.3 mRNA levels detected by using quantitative real-time PCR were increased by GDF-15 for reasons that are unclear. Cav1.2 and Cav1.3 are encoded by the cacna1C and cacna1D genes respectively [v1.2 and Cav1.3 channels differ according to interaction with different intracellular proteins [v1.2 and PDZ (PSD-95/Dlg/ZO1) domain proteins plays an important role in coupling L-type Ca2+ channel activity with the phosphorylation of nuclear CREB (cAMP-response-element-binding protein) [v1.3 with Shank results in its targeting to phosphorylated (p)CREB at synapses [v1.3 have also been reported in which the C-terminal modulatory domain confers unique gating properties [v1.2 and Cav1.3 protein expression by GDF-15 is due to variation in protein structure or a post-transcriptional mechanism remains an open question.Cafunction ,43. We oectively . The regproteins ,46. For protein) , whereassynapses ,48. Struoperties ,50. Whetv2.1 expression and IK [v2.1 expression may be exerted via TβRII-induced activation of Src [v1.3 expression induced by GDF-15 is required for the activation of TβRII and PI3K/Akt/mTOR signalling pathways, confirming our previous finding of a non-Smad mechanism [v1.3 expression, suggesting that activation of the ERK pathway is required for this effect. A previous study showed that ERK activation regulates Kv4 channel subunits at the transcriptional and post-translational levels [+ channels, as in the regulation of IK by growth factors [ICa nor has an immediate effect on Ca2+ amplitude (results not shown), instead it increased the mRNA level of Cav1.3 and luciferase expression driven by Cav1.3 promoter, we believe that the activation of ERK signalling by GDF-15 regulates Cav1.3 expression at the transcriptional level.Our previous study found that Akt/mTOR and MAPK/ERK pathways were activated in CGNs by GDF-15 treatment, consistent with findings in non-neuronal cells ,52, althl levels . ERK can factors . Since G2+ was known to play a critical role in coupling neuronal activity to gene transcription. Ca2+ influx via postsynaptic L-type Ca2+ channels activates pCREB [v1.3 translocates to the nucleus where it functions as a transcriptional regulator to modulate the transcription of Ca2+-activated K+ channels in atrial myocytes [v1.3−/− mice have implicated Cav1.3 channels in auditory brainstem physiology and development [v1.3 channels; for instance, Cav1.3 channel deficiency reduces long-term fear memory, antidepressant-like behaviour and congenital deafness [Neuronal L-type Caes pCREB ,48 and Nes pCREB , which smyocytes , and stuelopment ,58. Varideafness –61. In c


The corresponding changes in the fluorescence are shown in C and D, respectively.Trace (A) shows a typical transient [Ca •2+]i oscillations.Supraoptic fluorescent vasopressin (AVP-eGFP) and oxytocin (OT-mRFP1) neurones exhibit distinct spontaneous [Ca•2+]i oscillations, intensifies existing oscillations, and exceptionally stops oscillations.Vasopressin triggers i oscillations. In the neurones identified (through specific fluorescence), about 80% of AVP neurones and about 60% of OT neurones exhibited these oscillations. Exposure to AVP triggered [Ca2+]i oscillations in silent AVP neurones, or modified the oscillatory pattern in spontaneously active cells. Hyper- and hypo-osmotic stimuli (325 or 275 mOsmol/l) respectively intensified or inhibited spontaneous [Ca2+]i dynamics. In rats dehydrated for 3 or 5 days almost 90% of neurones displayed spontaneous [Ca2+]i oscillations. More than 80% of OT-mRFP1 neurones from 3 to 6-day-lactating rats were oscillatory vs. about 44% (OT-mRFP1 neurones) in virgins. Together, these results unveil for the first time that both AVP and OT neurones maintain, via Ca2+ signals, their remarkable intrinsic in vivo physiological properties in an isolated condition.The magnocellular vasopressin (AVP) and oxytocin (OT) neurones exhibit specific electrophysiological behaviour, synthesise AVP and OT peptides and secrete them into the neurohypophysial system in response to various physiological stimulations. The activity of these neurones is regulated by the very same peptides released either somato-dendritically or when applied to supraoptic nucleus (SON) preparations  These hormones are released from axonal projections from these neurones to the neurohypophysis into the portal blood circulation in response to various physiological stimuli such as dehydration, osmotic stimulation, parturition and lactation. Both AVP and OT neurones exhibit characteristic electrical activities. Under physiological settings, the AVP neurones exhibit ‘phasic’ firing activity of action potentials with intervals, whereas OT neurones fire high frequency synchronized bursts of action potentials associated with suckling-induced milk ejection. The characteristic firing patterns, as recorded in vivo experiments, the somato-dendritic release of AVP modulates the phasic pattern of AVP neurones electrical activity depending on the initial firing pattern 2+ concentration ([Ca2+]i) in isolated AVP-containing neurones 2+]i responses are modulated by specific voltage-gated Ca2+ channel subtypes 1a, V1b and V2) 2+]i in isolated OT-containing neurones through the activation of specific OT receptors and the release of Ca2+ from thapsigargin-sensitive intracellular stores 2+ entry 2+]i responses induced by AVP critically require plasmalemmal Ca2+ influx, indicating the primary role for membrane Ca2+ channels. Further studies have shown that the existence of a cell specific (AVP and OT) Ca2+ homeostasis and Ca2+ clearance mechanisms depends upon physiological conditions related to the specific electrical firing patterns of these neurones In the 2+]i responses to AVP or OT), displayed spontaneous [Ca2+]i oscillations under normal experimental conditions 2+]i oscillations were clearly established.The changes in AVP release induced in response to plasma osmolarity fluctuations are mediated by regulation of action potential discharge in the cell bodies 2+]i dynamics in i) AVP-sensitive and OT-sensitive (in terms of their specific [Ca2+]i responses) neurones from wild type adult male and virgin Wistar rats; and ii) identified AVP and OT neurones from homozygous transgenic male, as well as from virgin and lactating Wistar rats models expressing (1) an arginine vasopressin (AVP)-enhanced green fluorescent protein (AVP-eGFP) In the present study, we studied i in isolated SON neurones was measured with a fluorescent Ca2+ indicator Fura-2 according to the procedure previously described 2+]i measurements: a fluorescence microspectrofluorimetry system  for single-detector experiments and an imaging system using a CCD camera as a detector for video imaging and for measurement of [Ca2+]i from neurones obtained from transgenic animals.The [Ca2.4This system is based on an epi-fluorescence inverted microscope . The excitation light from a Xenon lamp passed through bandpass filters mounted on a computer-controlled rotating wheel that allows alternate stimulation at 340 ± 10 and 380 ± 10 nm with a frequency of 3.3 Hz. The excitation light was deflected by a dichroic mirror  through an oil-immersion objective . Fluorescence emission from individual cells was spatially limited by a diaphragm adjusted to the cell size (10–15 μm).2.52+]i was performed using an Axio Observer D1 (Zeiss) inverted microscope equipped with filters for monitoring GFP and RFP fluorescence, and epifluorescence oil immersion objectives . This allowed us to visualize and identity the SON neurones obtained from AVP-eGFP and OT-mRFP1expressing animals. The excitation light from a Xenon lamp passed through a Lambda D4 ultra-fast wavelength switching system (Sutter Instruments) with a maximum switching frequency of 500 Hz. The fluorescence intensity was detected by using a cooled CCD camera  and the whole system was controlled by Zeiss ZEN Imaging software (2012-SP2/AxioVision SE64 Rel. 4.8.3). The fluorescence intensity was measured with excitations at 340 and 380 nm, and emission at 510 nm.Video imaging of [Ca2+]i in nM a calibration was performed on a few neurones for both systems. An estimation of [Ca2+]i was determined from the f340/f380 ratio using the Grynkiewicz equation min = 0.24, Rmax 4.66, β = 3.39. Calibration performed with the imaging system gave Rmin = 0.2, Rmax = 7.2, β = 7.7. The dissociation constant for Fura-2 at 37 °C was assumed as KD = 224 nM.To estimate i trace was first classified as oscillating or non-oscillating. Changes in [Ca2+]i with an amplitude of at least 30 nM that occurred within 120 s (or faster) were considered to be “Ca2+ events”, and traces or parts of traces in which such events occurred repeatedly were considered “[Ca2+]i oscillations”. The threshold of 30 nM was used as a value sufficient for excluding the interference of the noise inherent in the signal. Only [Ca2+]i traces with sustained oscillations lasting for at least 10 min were included in further analysis. Traces with strong trend or obvious artefacts were removed from the data set. To reduce noise, all data were filtered by a 2nd-order Butterworth low-pass filter with a cut-off frequency of 0.5 Hz (with filter coefficients estimated by the MATLAB function butter and filtration performed by filtfilt). MATLAB 2015a and its Statistics, Machine Learning, and Signal Processing toolboxes were employed in all numerical procedures.Each [Ca2+]i. We estimated this trend by averaging 55 traces with a duration of more than 15 min and fitting the averaged trace with a linear function of time. This gave a trend expressed as 0.011 nM/s . This increase was then subtracted from all individual traces.Although ratiometric measurements using Fura-2 reduce the effect of photo bleaching, some trend of it exists, especially in longer-lasting experiments. This is because signals from 340 nm and 380 nm do not bleach equally and the ratio is rising even without a change in [Ca2.82+]i baseline, the mean [Ca2+]i level, the standard deviation of the trace, and the skewness of the [Ca2+]i values. These parameters were computed separately for each trace segment corresponding to a defined physiological condition . Only trace segments longer than 300 s were considered. An example of such evaluation is shown in Four basic parameters were computed for each individual trace or its segment: the [Ca2+]i baseline 2+]i values in each trace or trace segment. The mean [Ca2+]i level was calculated as the average of all [Ca2+]i values. i.e., 2+]i samples in the trace segment and The [Ca2+]i values within the trace segment. i.e., 2+]i values within the trace. The asymmetry of this spread is quantified by the distribution skewness 2+]i values. For a symmetric distribution , The standard deviation t-test was used to determine if the difference was statistically significant. The p-values reported in Results represent the significance level for rejecting the null hypothesis of zero difference. The MATLAB function t-test was used.Differences between the trace segments were evaluated based on the four trace parameters defined above. For each parameter, the difference of its value before and after drug application was calculated for each cell trace .In all [Ca33.12+]i oscillations in 79 out of 112 unidentified neurones (71%). Among identified neurons, 12 out of 15 AVP cells (80%) and 9 out of 15 OT cells (60%) exhibited oscillations. 2+]i oscillation in an identified AVP-eGFP neurone (A) and in OT-mRFP1 neurone (B). To compare the course of spontaneous oscillations vs. an evoked transient, we show in 2+]i response induced by 50 mM K+ observed in an AVP-eGFP neurone, while 2+]i oscillations observed in an AVP-eGFP neurone. The corresponding spatial changes in the fluorescence are shown in We observed spontaneous i oscillations was highly heterogeneous, and can be qualitatively classified into the following three types. The type I pattern is characterized by Ca2+ events of regular amplitude and frequency , we sought a simple quantitative classifier that would allow us to systematically express such pattern changes. The frequency or amplitude of the oscillation peaks could not be used for this purpose, as these parameters become ill-defined for irregular oscillations C. We fou I as in A, modera I as in B, and sy I as in C. The stγ=0.5 in F. Fig. 43.22+]i dynamics in oscillating neurones was monitored for 300 s in NL with a standard osmolarity of 295–300 mOsm/l. Then the perfusion solution was switched to either hypo-osmotic (275 mOsm/l) or hyper-osmotic (325 mOsm/l) solution and [Ca2+]i dynamics was monitored for another 300 s. Representative examples of oscillating neurones subjected to hyper- (A) or to hypo- (B) osmotic stimuli are shown in To evaluate the effect of osmolarity, [Cavs. hypo- or hyper osmolarity condition was statistically significant  and in the spread 2+]i within the trace segment . The other two evaluated parameters, the [Ca2+]i baseline and the skewness of the [Ca2+]i distribution, did not change significantly (p = 0.82 and 0.08). For neurones exposed to hyper-osmotic solution (n = 48), the increase of mean [Ca2+]i level was 11.8 ± 5.1 nM and the increase of p-value 0.02), while the changes in baseline and skewness were not (p = 0.1 and 0.9).For each neurone, we calculated the four basic trace parameters, and evaluated if the change in these parameters in normal i.e., the state in the normal osmotic condition) was taken into account. The skewness of the [Ca2+]i distribution tends to increase in neurones that had low initial value of skewness, and to decrease in neurones that had high initial skewness  of [Ca2+]i baseline with an average change of 13.4 ± 3.2 nM. In addition, AVP elicited very significant (p = 0.001) negative change in the skewness of the [Ca2+]i distribution giving a strong average decrease of 1.26 ± 0.27. When the initial skewness was in the intermediate range (type II oscillation), it was only weakly affected by AVP, while in neurones with high initial skewness (type III), AVP typically prompted a switch to type II oscillatory behaviour .Three groups of animals were prepared. In the control group, the rats were maintained under normal conditions , was significantly increased compared to the OT-mRFP1 neurones from transgenic adult virgin rats: 7 of 16 (44%). However, no significant difference was detectable among the patterns of oscillations in these groups of neurones .We also measured the [Ca42+]i oscillations of SON AVP and OT neurones in isolated condition, and show how these oscillations are affected by the physiological state of the animal  and by exposure to extracellular stimuli . The majority of AVP neurones (about 80%) and about 60% of OT neurones were oscillatory. Under lactating conditions, there was a significant increase in the number of oscillatory OT neurones (from 44% to about 80%). Spontaneous [Ca2+]i oscillations exhibited a wide range of patterns, ranging from regular oscillations with stereotypical [Ca2+]i peaks to irregular fast oscillations (Section 2+]i dynamics (present study). Given the heterogeneous character of the oscillations, their consistent characterization by frequency and amplitude was not feasible. Instead, we relied on a combination of four quantitative parameters: the [Ca2+]i baseline, the mean [Ca2+]i level, the spread (standard deviation) of [Ca2+]i values, and the skewness of the [Ca2+]i distribution. The skewness parameter, which quantifies the asymmetry of the distribution of [Ca2+]i values, was used to classify the oscillation patterns .It has been known that SON neurones are osmosensitive and that their behaviour, notably, the electrical activity of AVP neurones acutely isolated from the rat supraoptic nucleus, is increased by hypertonicity and inhibited by hypotonicity in the absence of neighbouring glial cells and without any synaptic connectivity The effects of dehydration on AVP and OT neuronal activity, as well as on release of AVP and OT at the level of soma and neurohypophysis have been widely discussed 2+]i oscillations. In previous studies, using in vivo electrophysiology and push-pull techniques, the somatodendritic release of AVP was shown to modulate electrical activity of magnocellular neurones in vitro release at the level of neurohypophysis or from isolated SON neurones 2+]i oscillations observed in AVP neurones similarly display several patterns and AVP modulated these oscillations in a likewise manner: in silent neurones, AVP triggered the oscillations; in oscillating neurones, AVP intensified these oscillations, and in a rare case, AVP attenuated a pronounced oscillation.Another aspect of our study was in dissecting various effects of AVP on the spontaneous [Ca2+ signals. OT-induced [Ca2+]i responses are not only modulated by OT receptors but also OT activates thapsigargin-sensitive intracellular Ca2+ stores in vivo experiments, or in slice preparations, OT always showed an excitatory/positive and synchronised effect on their firing behaviour, leading to the release of a bolus amount of OT to meet their end functions. In our experiments, about 60% of OT neurones showed spontaneous oscillations in [Ca2+]i (44% in identified OR-mRFP1 neurones from virgins), these neurones were osmosensitive and sensitive to dehydration. More strikingly, nearly 80% of OT neurones from lactating rats became oscillatory. This finding suggests the possibility, as of in vivo effects, that almost all OT neurones should exhibit spontaneous [Ca2+]i oscillation, which would facilitate a massive release of OT during lactation vs. non-lactating/virgin state or in males. However, further experiments are necessary to identify whether spontaneous [Ca2+]i oscillations of all OT-mRFP1 neurones are synchronised during lactation, as well.The autoregulatory mechanism of OT neurones has been clearly established 52+]i oscillations in an isolated condition under various experimental conditions. These properties are shown to mimic their intrinsic electrical behaviour under physiological conditions such as osmotic shock, dehydration and lactation. The understanding of autoregulatory mechanisms of AVP and OT neurones 2+]i activity.In the present paper we show, for the first time, that fluorescent magnocellular supraoptic AVP-eGFP and OT-mRFP1 neurones exhibit distinct spontaneous [CaThe authors state that they have no conflict of interest pertaining to this manuscript.SK, CS, OF, GD: performed experiments.YU, OF: prepared and maintained the heterozygous and homozygous transgenic rats for both AVP and OT and developed double transgenic rats for AVP and OT; performed genotyping, manuscript writing.SK, MZ, AV, GD: data and statistical methods and analysis; manuscript writing.AV, GD: prepared the concept of the project.AC, ES, GD: project management and logistics, manuscript writing.


CD38-dependent cyclic ADP-ribose (cADPR) is also involved in this autoregulation by elevating [Ca2+]i via Ca2+ mobilization through ryanodine receptors on intracellular Ca2+ pools that are sensitive to both Ca2+ and cADPR. In addition, it has recently been reported that heat stimulation and hyperthermia enhance [Ca2+]i increases by Ca2+ influx, probably through TRPM2 cation channels, suggesting that cADPR and TRPM2 molecules act as Ca2+ signal amplifiers. Thus, OT release is not simply due to depolarization–secretion coupling. Both of these molecules play critical roles not only during labor and milk ejection in reproductive females, but also during social behavior in daily life in both genders. This was clearly demonstrated in CD38 knockout mice in that social behavior was impaired by reduction of [Ca2+]i elevation and subsequent OT secretion. Evidence for the associations of CD38 with social behavior and psychiatric disorder is discussed, especially in subjects with autism spectrum disorder.Oxytocin (OT) is released into the brain from the cell soma, axons, and dendrites of neurosecretory cells in the hypothalamus. Locally released OT can activate OT receptors, form inositol-1,4,5-trisphosphate and elevate intracellular free calcium (Ca Oxytocin (OT) and arginine vasopressin (AVP) are nonapeptides that differ in two amino acid residues . OT and OT, AVP, and their receptors are present in the brain not only in females during specific reproductive periods but also in non-reproductive females and males . AccumulAnother reason to focus on the release is that the mechanism contains a very important aspect in terms of physiological science, in that the proposed idea challenges the principal rule in physiology of depolarization–secretion coupling –24. FurtOT is secreted from the nerve terminals of axons of oxytocinergic neurons at the perivascular site in the posterior lobe of the pituitary into the circulation  Fig. 1)1). OxytoLocally released OT causes excitation of OT neurons by activating OT receptors expressed in neurons of both the PVN and SON –43. OT sq/11-type GTP-binding protein i increases: a rapid initial increase and a sustained elevation lasting for 5 min [2+]i, and this phase is IP3-dependent. Pretreatment with 8-bromo-cADPR, an antagonist of the cADPR-binding site of Ca2+ release channels of ryanodine, inhibits OT-mediated sustained [Ca2+]i increases. ADPR and β-NAD+ also induce elevation of [Ca2+]i and replicate the second phase of sustained [Ca2+]i increases i shows little change in either phase, suggesting that the two phases of [Ca2+]i elevation in hypothalamic neurons are due to Ca2+ mobilization from the intracellular Ca2+ pools i signaling via CD38 or TRPM2 channels downstream of OT receptors. [Ca2+]i increases in the model neuron, NG108-15 mouse neuroblastoma × rat glioma hybrid cells that possess CD38 [2+]i elevation was observed at 40 °C in the absence of extracellular Ca2+. Ca2+ influx is expected, probably through non-selective cation TRPM2 channels, because elevation of [Ca2+]i is inhibited by the TRPM2 channel inhibitor, 2-aminoethoxydiphenyl borate (2-APB). Similarly, 8-bromo-cADPR inhibits responses to β-NAD+ and heat. These results suggest that cADPR contributes to both Ca2+ mobilization from internal Ca2+ pools and Ca2+ influx through TRPM2 Ca2+-permeable channels from the extracellular space. Such [Ca2+]i increases may result in OT release. However, there have been no previous reports regarding heat-induced OT release in the hypothalamus.Extracellularly applied cADPR can activate i. This hypothesis of depolarization-independent but heat-sensitive Ca2+ signaling for OT release is consistent with the previous suggestion of dendritic release of OT without depolarization [This review discussed how OT is released into the brain. Camus Fig. . In addirization , 21, 39.OT exerts an anxiolytic effect during stress, and stress sometimes induces hyperthermia. It is therefore interesting to examine how stress induces hyperthermia, which results in subsequent OT release. OT release seems to be important in damping the stress-induced disadvantage.2+ signaling and OT transport from the blood to the brain remain to be resolved.OT is an essential molecule for social memory and social behavior , 29. Def


By contrast, sustained global cytosolic Ca2+ elevations decrease ATP levels and cause necrosis, leading to the disease acute pancreatitis (AP). Toxic Ca2+ signals can be evoked by products of alcohol and fatty acids as well as bile acids. Here, we have investigated the mechanism by which l-asparaginase evokes AP. Asparaginase is an essential element in the successful treatment of acute lymphoblastic leukaemia, the most common type of cancer affecting children, but AP is a side-effect occurring in about 5–10% of cases. Like other pancreatitis-inducing agents, asparaginase evoked intracellular Ca2+ release followed by Ca2+ entry and also substantially reduced Ca2+ extrusion because of decreased intracellular ATP levels. The toxic Ca2+ signals caused extensive necrosis. The asparaginase-induced pathology depended on protease-activated receptor 2 and its inhibition prevented the toxic Ca2+ signals and necrosis. We tested the effects of inhibiting the Ca2+ release-activated Ca2+ entry by the Ca2+ channel inhibitor GSK-7975A. This markedly reduced asparaginase-induced Ca2+ entry and also protected effectively against the development of necrosis.Exocytotic secretion of digestive enzymes from pancreatic acinar cells is elicited by physiological cytosolic Ca2+ and ATP together to control life and death’.This article is part of the themed issue ‘Evolution brings Ca Gerasimenko et al. i owing to release of Ca2+ from internal stores, followed by Ca2+ entry from the extracellular solution. The sustained [Ca2+]i elevation reduces ATP formation. These effects can be markedly reduced by specific pharmacological blockade of CRAC channels, which also markedly reduces the extent of necrosis.Our results show that asparaginase acts on protease-activated receptor 2 (PAR2) to evoke sustained elevations of [Ca2.(a)2+]i in isolated acinar cell clusters causing intracellular Ca2+ overload. This is owing to Ca2+ release from internal stores triggering excessive store-operated Ca2+ entry [2+]i over a wide concentration range. One of the challenges inherent in this approach is that the time course needed for a study on normal freshly isolated cells is quite different from that of the development of AAP in the clinical situation. AAP typically develops several days after several injections (over many weeks) of asparaginase i oscillations (9 out of 42 cells). Figure 1a shows a representative positive trace with repetitive Ca2+ spikes induced by 20 IU ml−1, whereas figure 1b represents the more typical negative response (33 out of 42 cells), in which the same concentration of asparaginase failed to cause any change in [Ca2+]i. A higher concentration of asparaginase (200 IU ml−1) elicited repetitive [Ca2+]i oscillations, mostly on top of a sustained elevated [Ca2+]i . The development of a sustained [Ca2+]i elevation has previously been shown to be a distinctive characteristic of [Ca2+]i changes induced by pathological concentrations of alcohol metabolites or bile acids [2+]i plateau, although often small, was seen in the vast majority of cells (52 out of 55) stimulated by asparaginase (200 IU ml−1). In some cases (12 out of 55), there were no, or very few, spikes superimposed on the elevated [Ca2+]i plateau (figure 1d).We found that only in some cells did a low concentration of asparaginase 2+ always terminated the elevated [Ca2+]i plateau , but did not significantly (p > 0.27) change the amplitudes of the asparaginase-induced Ca2+ spikes (n = 28) within the time frame of our experiments (e). Ca2+ entry clearly plays an important role in the formation of the asparaginase-induced elevated [Ca2+]i plateau and the role of Ca2+ entry was further demonstrated by increasing the external Ca2+ concentration to 2 mM during asparaginase stimulation, which caused a marked and sustained increase in [Ca2+]i, . Subsequent removal of external Ca2+ and addition of the Ca2+ chelator EGTA (200 µM) abolished the elevated [Ca2+]i plateau, whereas the Ca2+ oscillations continued for some time (f).Removal of external Ca plateau c,d, but eriments e. Ca2+ e n = 18; f. Subseqome time f.2+ entry into pancreatic acinar cells induced by alcohol metabolites or bile acids, as well as their pathological effects, can be markedly inhibited by the CRAC channel blockers GSK-7975A and CM-128 [2+]i elevation and found that it was abolished in the presence of the inhibitor, although repetitive Ca2+ spiking was still observed within the time frame of our experiments . Figure 1h summarizes the degree of inhibition, caused by removal of external Ca2+ or by GSK-7975A, of the integrated Ca2+ signal evoked by asparaginase.It has previously been shown that the excessive Cad CM-128 ,52. We heriments g, n = 32(c)2+ release channels in asparaginase-induced [Ca2+]i elevations, we have used caffeine, a substance known to reliably inhibit IP3-mediated intracellular Ca2+ release in pancreatic acinar cells [2+]i elevations in a Ca2+-free solution . The phospholipase C (PLC) inhibitor U73122 (10 µM) also significantly blocked the asparaginase-induced Ca2+ release as well as the response to 1 µM ACh . Ryanodine (100 µM), inhibiting RyRs, also substantially reduced the asparaginase-induced [Ca2+]i elevations .To investigate the involvement of intracellular Caar cells . Caffein−1)-induced [Ca2+]i elevations were profoundly inhibited , whereas ACh (1 µM), eliciting Ca2+ release via IP3Rs independently of NAADP [2+]i rise .It has previously been shown that NAADP signalling in pancreatic acinar cells can be inactivated by the cell-permeable NAADP analogue, and selective antagonist, Ned-19 . After pof NAADP , was sti(d)3 and diacylglycerol [2 (10 µM) before the addition of asparaginase (200 IU ml−1). The PAR2 blocker reduced significantly  the asparaginase-induced [Ca2+]i oscillations as well as the sustained [Ca2+]i elevation .PAR2 is widely expressed in human and animal tissues, including the pancreas, and has previously been implicated in the pathology of AP ,54. The glycerol . Therefolevation e; n = 32(e)2+ movements in more detail, we have applied a specific protocol routinely used to assess Ca2+ entry and extrusion i change (b) and the rate of [Ca2+]i increase . However, the quantitatively most important effect of asparaginase was to slow down the rate of Ca2+ extrusion after removal of external Ca2+ (figure 3a). The half-time of [Ca2+]i recovery was more than three times longer than in the control cells (figure 3d).To study Caxtrusion ,38. In t removed a. In thencreased a, assessi change b and the(f)2+ extrusion is an energy-demanding process and has previously been found to be abnormal in pancreatic acinar cell pathologies owing to disruption of mitochondrial metabolism and, therefore, reduction of ATP levels [2+]i i –58. By toacetate .Figure 2+ was important for the cytoplasmic and mitochondrial effects induced by asparaginase, we decided to check if inhibition of Ca2+ entry channels i elevations (e) and the asparaginase-induced necrosis (b), suggesting that PAR2 inhibitors in addition to, or in combination with, CRAC channel inhibitors could be a useful tool to supplement asparaginase ALL treatment in AAP cases.The mechanism by which asparaginase induces pancreatitis is fundamentally different from its therapeutic action on the lymphoblastic cells in ALL. The asparaginase effect on cancer cells relies on depletion of asparagine, which the malignant cells cannot produce themselves, in contrast to normal cells , whereasbly TPCs . The aspbly TPCs d. This mraginase , withoutevations e and thenecrosis b, sugges2+ entry and extrusion are significantly affected by asparaginase, leading to formation of the pathological elevated [Ca2+]i plateau, and this sustained elevation of [Ca2+]i would appear to be responsible for the necrosis. The demonstrated reduction of the intensity of Ca2+ extrusion (a) is clearly a key element, and the simplest explanation for this is the reduction in the intracellular ATP level (a) limiting the energy supply to the Ca2+ ATPase in the plasma membrane. When energy supply is partially restored by the addition of pyruvate (d), it provides a similar degree of protection against pancreatic necrosis to PAR2 inhibition or GSK-7975A (b). Clearly, both Ca2+ and ATP play key roles in pancreatic pathology, as indeed they do in physiological regulation of secretion, and therapeutic strategies must take both into account.Both Caxtrusion a is cleaTP level a limitinpyruvate d, it proSK-7975A b. Clearl4.(a)All fluorescent dyes were purchased from ThermoFisher Scientific , and CPA was from Merck Millipore . Collagenase was obtained from Worthington (USA), asparaginase was from Abcam (UK), the PAR2 inhibitor FSLLRY-NH2 from TOCRIS (UK) and GSK-7975A from GlaxsoSmithKline (UK). All other chemicals were purchased from Sigma. C57BL/6 J mice were from Charles River UK Ltd.(b)−1, Worthington, UK) and incubated in a 37°C water bath for 14–15 min. The extracellular solution contained: 140 mM NaCl, 4.7 mM KCl, 10 mM Hepes, 1 mM MgCl2, 10 mM glucose, pH 7.2, and CaCl2 (0–2 mM as described in the text).Pancreatic acinar cells were isolated as previously described . Briefly(c)2+]i, isolated pancreatic acinar cells were loaded with Fluo-4-AM  or Fura-2-AM  following the manufacturer's instruction. The cells were then washed and re-suspended in extracellular solution containing 1 mM CaCl2.For measurements of [Ca2+]i at concentrations around the resting [Mg2+]i (approx. 0.9 mM). This was used as an indirect approach to detect cytosolic ATP depletion [Measurement of intracellular ATP was performed with MgGreen, which senses changes in [Mgepletion . Pancreaepletion .Necrotic cell death was assessed with PI uptake as previously described .All experiments were performed at room temperature using freshly isolated cells attached to coverslips of the perfusion chamber. Fluorescence was imaged over time using an Leica TCS SPE confocal microscope.(d)p-values were calculated using t-test or ANOVA, with p < 0.05 considered significant.Data are presented as mean ± SEM. Statistical significance and


Here, we report a thorough theoretical and experimental characterization of myosin-derived SHG intensity profiles within intact zebrafish skeletal muscle. We determined that “SHG vernier” patterns, regions of bifurcated SHG intensity, are illusory when sarcomeres are staggered with respect to one another. These optical artifacts arise due to the phase coherence of SHG signal generation and the Guoy phase shift of the laser at the focus. In contrast, two-photon excited fluorescence images obtained from fluorescently labeled sarcomeric components do not contain such illusory structures, regardless of the orientation of adjacent myofibers. Based on our results, we assert that complex optical artifacts such as SHG verniers should be taken into account when applying functional SHG imaging as a diagnostic readout for pathological muscle conditions.SHG microscopy is an emerging microscopic technique for medically relevant imaging because certain endogenous proteins, such as muscle myosin lattices within muscle cells, are sufficiently spatially ordered to generate detectable SHG without the use of any fluorescent dye. Given that SHG signal is sensitive to the structural state of muscle sarcomeres, SHG functional imaging can give insight into the integrity of muscle cells  SHG-derived contrast has a particularly attractive property relevant to biological imaging. The signal is tunable, meaning that specific illumination wavelengths can be chosen to minimize (i) background photons  as well as (ii) spectral overlap with other contrast agents being imaged in parallel 345Second harmonic generation (SHG) is a nonlinear optical process where two photons interact with a highly ordered, non-centrosymmetric material  to generate a single photon with half the wavelength or double the frequency of the incident photons1To produce detectable amounts of SHG signal, the SHG photons generated by different molecules must be in phase, leading to constructive interference of the signal. Certain components such as collagen, microtubules, and muscle myosin arrays possess sufficient order to fulfill these strict symmetry constraints. These biological structures generate detectable SHG signal, enabling them to be imaged in a label-free manner7The coherent nature of SHG has proven especially relevant when visualizing myosin bundles within the sarcomeres of striated muscle11Rana temporaria frog muscle fibers in the late 1950s121516One prominent SHG signal motif that is common in endogenous SHG imaging of skeletal muscle in both the zebrafish and the mouse are so-called “SHG verniers” — ‘Y’-shaped patterns and curved distortions in SHG signal between sarcomeric units in a myofiber . These vin silico. In this model, we manipulated the geometry of adjacent synthetic muscle cells to determine the possible SHG signal patterns that may arise when illuminated with focused laser light (see Methods). To act as a standard for comparison of SHG signal from the muscle myosin, we also added a fluorescent component to the model, specifically considering the myosin thick filaments to be fluorescently labeled in addition to being SHG capable. The simplified hypothetical muscle tissue in our model is made up of two adjacent myofibers with multiple sarcomeric units . Additionally, we assumed an NA of 0.8 for the objective and 0.55 for the condenser in our simulations, as these are reasonably standard values when performing high resolution laser scanning imaging in many biological applications.To investigate the source of SHG vernier patterns in muscle tissue, we constructed a simplified numerical simulation of an SHG microscopy experiment. We first applied our theoretical characterizations by modeling hypothetical muscle fibers ic units  organizeOur calculations yielded striking differences in TPEF  and SHG If verniers were in fact optical artifacts of the SHG process, we should be able to see differing signal in staggered muscle fibers when comparing SHG and TPEF images. Fluorescent emission from fluorescently labeled sarcomere components would show no increased fluorescence between fibers and would not have curvature or bifurcations in the signal, while SHG curvature and verniers would be prominent in the same imaging plane , dependiSince the model predicts that illusory SHG verniers have the potential to cross the plasma membrane boundaries of adjacent muscle cells . In this case, the myosin-derived SHG co-localizes with the TPEF from the fluorophore-conjugated secondary antibody . In long10Gt(tpm3-citrine)ct5a established by Trinh et al.In a second experimental condition, we wanted to ensure that the SHG verniers that we had seen were not an artifact of the myosin antibody labeling. In an effort to visualize labeled thin filaments within sarcomeres, we took advantage of the FlipTrap transgenic line To account for the complex endogenous SHG signal patterns that arise in whole zebrafish tissue, we expanded our initial simplified SHG model into arbitrary geometries. We aimed to recapitulate vernier patterns in real tissue, validating the expected differences in TPEF versus SHG signal seen in the original theoretical analysis . Our samWithin this volume, vernier pattern signal in the endogenous SHG channel  crosses In an effort to uncover the source of SHG verniers within intact muscles, we implemented a theoretical model, paying special attention to the consequences of the phase coherence of SHG propagation and the Guoy phase shift of a laser at the sample focus. Unlike fluorescent signal within the sarcomere, which always retains a banded pattern that is evenly spaced and unconnected, SHG signal patterns can be distorted, depending on the orientation of the incoming laser as well as the organization of adjacent muscle fibers. Our analysis of phase coherence and cancellation proved to be invaluable for discovering this optical artifact. Comparable phase considerations have benefited other nonlinear microscopic approaches, such as coherent anti-Stokes Raman scattering microscopy27Although our model predicts the occurrence of SHG verniers when adjacent sarcomeres are staggered, we do not rule out the possibility that some fraction of vernier patterns are a result of physical remodeling processes in the muscle cells, as has been previously shown in isolated rat and Xenopus muscle tissueZebrafish were raised, hatched, injected, and maintained in a house colony as previously describedGt(tpm3-citrine)ct5a (Danio rerio) embryos were obtained by incrossing males and females from the same genetic background. These embryos were allowed to develop for ~18–20 hours before being treated with 1 × 1-phenyl 2-thiourea  to inhibit pigment formation for the following few days of development. PTU medium was exchanged once every day or two. In the days before imaging, the embryos were kept in egg waterWild type (WT) or ine)ct5a  transgen2+/Mg2+-free 1× PBS 3 times . Gt(tpm3-citrine)ct5a zebrafish and the WT zebrafish were immediately prepared for imaging.To fix the zebrafish, the embryos/larvae were first anesthetized in 0.015–0.03% MS-222  and then placed in 4% methanol-free formaldehyde  on ice. After ∼5 min, embryos were allowed to continue to fix at room temperature on a nutator  for 1 hour before being washed in Ca2+/Mg2+-free) + 1% dimethyl sulfoxide (DMSO) + 1% bovine serum albumin (BSA) + 1.5% Triton X-100 detergent (referred to as PBSTB). Anti-sarcomere myosin  and anti-myomesin (mMaC Myomesin B4) primary antibodies were obtained from the Developmental Studies Hybridoma Bank at the University of Iowa. Primary antibodies were incubated on a nutator at room temperature in PBSTB with the whole-mount zebrafish for 2.25 hours (1:3 dilution). Embryos were washed 3 times (15 min each) in PBSTB before being suspended in PBSTB and treated with goat anti-mouse IgG secondary antibodies (1:200 dilution) conjugated to Alexa Fluor 488 nm . The embryos were incubated with the secondary antibody protected from light at room temperature on a nutator for 2.5 hours. After 3 washes (10 min each), the embryos were prepared for imaging.Embryos that were stained with antibody were first blocked overnight at 4 °C in 1× PBS  optical sections as images that are taken parallel to the long axis of the zebrafish, while “transverse” (x-z) optical sections are images that are taken perpendicular to the long axis of the zebrafish.Images were processed in Imaris , ImageJ (NIH), and Adobe Photoshop CS3 (Adobe Systems), where minor linear brightness/contrast adjustments and filtering  were performed.From the large 3D volume multichannel  datasets, sub-volumes were extracted that contained both vernier-pattern SHG signal and appreciable TPEF signal. All of the subsequent algorithms for segmentation and estimation of the surface normal vectors were performed using a script written in MATLAB . Implementation of the full 3D theoretical model to recapitulate the experimental SHG data using the sarcomeric TPEF signal was performed using a custom script in the open source Julia language . Poisson distributed background noise was simulated in the model data using a custom MATLAB script.Because SHG verniers are often found deep within the muscle compartments within zebrafish somites where light and antibody penetration is reduced, TPEF signal emanating from the immunostained sarcomeric components was low. The resulting low signal-to-noise ratio TPEF images were difficult to process using automatic segmentation algorithms, so we instead took advantage of a segmentation toolkit to perform manual segmentation of the dataHow to cite this article: Dempsey, W. P. et al. Determination of the source of SHG verniers in zebrafish skeletal muscle. Sci. Rep.5, 18119; doi: 10.1038/srep18119 (2015).


In vitro experiments indicate that mutations that cause dilated cardiomyopathy (DCM) uncouple this modulation, but this has not been demonstrated in an intact contractile system. Using a Ca2+-jump protocol, we measured the effect of the DCM-causing mutation ACTC E361G on the equilibrium and kinetic parameters of Ca2+ regulation of contractility in single transgenic mouse heart myofibrils. We used propranolol treatment of mice to reduce the level of troponin I and myosin binding protein C (MyBP-C) phosphorylation in their hearts before isolating the myofibrils. In nontransgenic mouse myofibrils, the Ca2+ sensitivity of force was increased, the fast relaxation phase rate constant, kREL, was reduced, and the length of the slow linear phase, tLIN, was increased when the troponin I phosphorylation level was reduced from 1.02 to 0.3 molPi/TnI . Native myofibrils from ACTC E361G transgenic mice had a 2.4-fold higher Ca2+ sensitivity than nontransgenic mouse myofibrils. Strikingly, the Ca2+ sensitivity and relaxation parameters of ACTC E361G myofibrils did not depend on the troponin I phosphorylation level . Nevertheless, modulation of the Ca2+ sensitivity of ACTC E361G myofibrils by sarcomere length or EMD57033 was indistinguishable from that of nontransgenic myofibrils. Overall, EC50 measured in different conditions varied over a 7-fold range. The time course of relaxation, as defined by tLIN and kREL, was correlated with EC50 but varied by just 2.7- and 3.3-fold, respectively. Our results confirm that troponin I phosphorylation specifically alters the Ca2+ sensitivity of isometric tension and the time course of relaxation in cardiac muscle myofibrils. Moreover, the DCM-causing mutation ACTC E361G blunts this phosphorylation-dependent response without affecting other parameters of contraction, including length-dependent activation and the response to EMD57033.Phosphorylation of troponin I by protein kinase A (PKA) reduces Ca Early studies showed that oponin I , troponioponin I , and theoponin C . This caoponin C . Transger stress .2+ sensitivity is independent of the level of troponin I phosphorylation; therefore, by analogy to S22/23A transgenic mice, they proposed that this uncoupling was necessary and sufficient to cause the DCM phenotype. It is uncertain whether uncoupling is also present in intact muscles that produce force, although indirect evidence from the ACTC E361G DCM mouse indicated a blunted response to β-adrenergic stimulation that was compatible with the uncoupling of the relationship between Ca2+ sensitivity and troponin I phosphorylation observed in vitro  in comparison with wild-type mice. We measured the effects of changing the [Ca2+], troponin I phosphorylation level, and sarcomere length (SL) on the isometric tension and relaxation rate after rapid Ca2+ jumps.Dilated cardiomyopathy (DCM) is a major cause of heart failure in humans, and a substantial proportion of cases of DCM are inherited. Mutations in the thin-filament proteins  that are associated with familial DCM have been studied particularly closely . By studin vitro . In this2+ sensitivity decreased 2- to 3-fold between 0% and ∼70% bisphosphorylation of troponin I and did not change at higher levels of phosphorylation  and the response to the Ca2+ sensitizer EMD57033.Recent studies have shown that Carylation . Since nrylation , the majrylation . TherefoACTC E361G transgenic mice  and non2 (0.5 mL/min) using a custom-made nose cone. A bolus of propranolol  was injected into the subclavian vein. The mice were kept in an anesthesia induction chamber for 30 min with 1.5% isoflurane and then sacrificed. The heart was removed and tissue samples (<5 mg) were dissected from the left ventricle, frozen, and kept in liquid nitrogen until myofibrils were prepared. Control samples were obtained from anesthetized mice that did not receive propranolol.Mice were anesthetized with 5% isoflurane  v/v in 100% oxygen (0.5mL/min), weighed, and then transferred to a heated surgical table  where anesthesia was maintained at 2.5% isoflurane v/v in 100% O2 1, EGTA 5, dithiothreitol (DTT) 5, NaN3 10, 2,3-butanedione-monoxime (BDM) 20, and 1% Triton X-100. All of our solutions contained the following protease inhibitors (μM): chymostatin 10, pepstatin 5, leupeptin 10, trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane) (E-64) 10, and phenylmethylsulfonyl fluoride (PMSF) 200. Triton X-100 and BDM were then removed from the permeabilized samples with a washing solution , and finally the samples were homogenized for 15 s with an Ultra-Turrax T10 blender  to produce a suspension of myofibrils. The suspension was washed two times by centrifugation and suspension in the washing solution. The final pellet was dissolved in 300 μL of washing solution and kept on ice for use within 3 days.Single myofibrils or thin bundles (two to seven myofibrils) were prepared from one piece of frozen heart sample by permeabilization and subsequent homogenization. Samples were immersed for 3 h in 2 mL of permeabilization solution containing (mM) Tris 10 (pH 7.1), NaCl 132, KCl 5, MgClACTC E361G propranolol-treated mice (15 weeks and 28 weeks old), and two from ACTC E361G-untreated mice (5 weeks and 28 weeks old)). Three to seven myofibrillar preparations were prepared for each experimental condition.In total, we used eight hearts (two from NTG propranolol-treated mice (4 and 28 weeks old), two from NTG-untreated (4 and 28 weeks old), two from μm/μN.We initiated contraction and relaxation using a fast-solution-change system and sensitive force transducer system, which were similar to those described previously . BrieflyWe achieved rapid activation and relaxation in myofibrils using an ultrafast-solution-change system constructed from a double-barreled micropipette mounted on a stepper motor that switched solutions in less than 10 ms. The micropipette was positioned perpendicular to the long axis of the myofibril. The relaxing and activating solutions were applied via adjacent barrels of the micropipette, with flow being driven by gravity. The stepper motor controlled the position of the micropipette relative to the myofibril, thus enabling changes between the two solutions, each flowing in a laminar pattern.2+]  controlled by a stepper motor was used to perfuse one barrel of the micropipette with a range of different activating solutions. We applied eight different solutions ranging from low to high [Ca2+] . The timμM Ca2+) and relaxing (0.01 μM Ca2+) solutions contained (mM) MOPS 10 (pH 7.0), MgATP 5, free Mg2+ 1, DTT 5, phosphocreatine 10, and creatine kinase (200 U/mL). The Ca-EGTA/EGTA ratio was set to obtain 10 mM total EGTA and the desired free [Ca2+]. Potassium propionate and sodium sulfate were added to adjust the ionic strength of the final solution to 200 mM. EMD57033 was dissolved in ethanol (30 mM) and then added in some experiments to activating and relaxing solutions at a concentration of 30 μM. Myofibrils were incubated in EMD57033-containing relaxing solution for 20–30 min at 17°C before measurements were started.Activating  was positioned horizontally between the microneedles described above and viewed with the video camera mounted on the microscope . The cross-sectional area was calculated from the observed diameter assuming a circular cross-section. Contraction and relaxation were initiated by the fast-solution-switch system described above. At each [Ca2+], a number of activation-relaxation cycles (range 1–5) were performed by the myofibril and the average value of measured parameters for the set of cycles was reported. Each contraction lasted for 2–5 s. If the myofibril sarcomere appearance or signal did not deteriorate after a series of contractions at short SL, the same myofibril was also used for measurements at long SL.A small droplet of myofibril solution was placed on the bottom of the glass chamber (temperature controlled to 17°C) and then the chamber was filled with relaxing solution. The selected myofibril or thin bundle (1–4 croscope . SarcomekACT) and fast phase of relaxation (kREL) were evaluated by curve-fitting using the Levenberg-Marquardt nonlinear least-square algorithm in LabVIEW.Apparatus control and data recording were done using a data acquisition device  and custom-written software in LabVIEW 2011 . The rate constants for exponential force development upon activation (2+] experiments were fitted to the Hill equation: y = y0 + Fmax[Ca2+]nH/(EC50nH + [Ca2+]nH) by adjusting the values of nH and EC50. Data were fitted and compared by means of an unpaired t-test using GraphPad Prism 6 . Best-fit parameters marked as ambiguous by GraphPad Prism were excluded from further analysis. In some cases, the bottom plateau (y0) was constrained to a constant value of 0.The maximum force was obtained by curve-fitting force development upon activation. The force data from the force- . The Ca2.0 ± 0.2 ).kACT, was not affected by phosphorylation, but changes in the relaxation parameters indicated that relaxation was faster in the phosphorylated myofibrils . Correspondingly, relaxation of ACTC E361G myofibrils was significantly slower than that of NTG myofibrils . There was no significant difference in the maximum force produced or the rate of force development ; p = 0.394). Similarly, there was no significant phosphorylation-dependent change in the time course of relaxation at fully activating [Ca2+] (p = 0.703 (tLIN), p = 0.912 (kREL); 2+ sensitivity in ACTC E361G myofibrils by a change in SL or addition of the Ca2+ sensitizer EMD57033 was similar to that observed for NTG myofibrils . Interestingly, the Ca2+-sensitizing effect was independent of the myofibril phosphorylation level, the SL, and the presence of the ACTC E361G mutation  specifically interferes with this modulation of Ca2+ sensitivity.We used the rapid CaThe troponin I of NTG mice hearts was phosphorylated at a level of 1.02 ± 0.03 mol Pi/mol (20% bisphosphorylation at Ser-22/23) in common with previous measurements . We achi2+ sensitivity occurs over the range of 0–70% occupancy of Ser-22 and Ser-23, with a relatively small decrease in Ca2+ sensitivity at higher levels of phosphorylation of troponin I  and troponin C is responsible for the higher Catroponin . We proptroponin . Modulatwitching .ACTC E361G mice had a substantially higher Ca2+ sensitivity of isometric tension , whereas with skeletal muscle troponin, ACTC E361G decreased Ca2+ sensitivity . In addC1 G159D , TPM1 D2M1 D230N , and TPMPM1 E54K .ACTC E361G is uncoupling, and not a change in Ca2+ sensitivity. Uncoupling is likely to be of physiological importance for the development of the DCM phenotype. Abolition of the normal response to β-adrenergic stimulation is expected to reduce cardiac reserve and predispose to heart failure , Ca2+ regulates kACT and isometric tension, but not tLIN or kREL; 2), changes in Ca2+ sensitivity are related to changes in tLIN or kREL, but not kACT; and 3), modulation of Ca2+ sensitivity by LDA is unrelated to modulation of Ca2+ sensitivity by phosphorylation changes or the ACTC E361G mutation.Since we measured the kinetics of Ca2+]; therefore, kACT is expected to be Ca2+ dependent, as shown in A and previously demonstrated for kACT and kTR . The relationship is not direct, since a 7-fold change in EC50 led to only a 2.7-fold change in tLIN and a 3.3-fold change in kREL. In contrast, kACT was not correlated with EC50. Previous measurements in murine myofibrils also showed a relationship between tLIN and kREL values and EC50 (We observed that changes in the myofilament Cag [Ca2+] B. The rand EC50 .2+ sensitivity is determined by the affinity of Ca2+ for troponin C in thin filaments (2+ sensitivity observed when cardiac troponin I is phosphorylated is linked to an increase in the rate of Ca2+ dissociation from troponin C in thin filaments (2+ sensitivity and the rate of relaxation.Cailaments . The decilaments , and thi2+ dissociating from troponin C coupled to the release of the C-terminus of troponin I from troponin C and its attachment to actin, where it blocks cross-bridge binding cooperatively (2+-free troponin switch (Relaxation is initiated by Caratively . The lagn switch .2+ dissociation from troponin C does not; therefore, it is possible for Ca2+ dissociation to become partly rate limiting at higher temperatures. According to the model of Stehle et al. (g, is given by the value of kACT when extrapolated to zero force. Plots of kACT versus force (gAPP is ∼2 s−1 independently of the phosphorylation level and the ACTC E361G mutation. Our tLIN and kREL measurements suggest that the time course of relaxation could be partly determined by the rate of Ca2+ dissociation in this system. Variations in the relative rates of cross-bridge detachment and Ca2+ dissociation in different experimental systems could determine whether or not the relaxation rate is related to Ca2+ sensitivity.Little et al.  pointed e et al. , the appus force  indicateACTC E361G.Myofibrils are the simplest contractile system in which LDA can be measured. In our myofibrils, we observed LDA of similar magnitude to that reported in skinned myocytes and muscle fibers, thus confirming that LDA is sarcomere based . It is i


Here we report the first solid-state fluorescent Ca2+ sensor that fulfills the essential requirements for realizing extracellular Ca2+ imaging. Inspired by natural extracellular Ca2+-sensing receptors, we designed a particular type of chemically-crosslinked polyacrylic acid gel, which can undergo single-chain aggregation in the presence of Ca2+. By attaching aggregation-induced emission luminogen to the polyacrylic acid as a pendant, the conformational state of the main chain at a given Ca2+ concentration is successfully translated into fluorescence property. The Ca2+ sensor has a millimolar-order apparent dissociation constant compatible with extracellular Ca2+ concentrations, and exhibits sufficient dynamic range and excellent selectivity in the presence of physiological concentrations of biologically relevant ions, thus enabling monitoring of submillimolar fluctuations of Ca2+ in flowing analytes containing millimolar Ca2+ concentrations.Although the role of extracellular Ca In living organisms, Ca2+ concentrations differ greatly depending on the compartment. Typically, the Ca2+ concentration is ~100 nanomolar (nM) in intracellular cytosol, ~100 micromolar (μM) in the endoplasmic reticulum and mitochondria and ~1 millimolar (mM) in extracellular fluid and blood  that are appropriate for the respective background Ca2+ concentrations. However, almost every Ca2+ indicator known to date has a Kd value ranging from nM to μM, and therefore allows for Ca2+ imaging only in cytosol and organelles  and high selectivity for Ca2+ in the presence of excessive amounts of other physiological ions. Although simple Ca2+ imaging against mM-order background concentration of Ca2+ may be possible using existing indicators with μM-order Kd, Ca2+ indicators with one-order higher Kd have a great advantage in monitoring Ca2+ transients and oscillations in extracellular regions. Even more challenging in extracellular Ca2+ imaging, one has to create a mechanism to avoid the outflow of indicators from an observation area through molecular diffusion. Obviously, this issue is intractable with existing molecular-based indicators.In fact, there are major problems in the development of indicators for extracellular Ca2+ sensor that can clear up all the above problems. It is a solid-state (gel) sensor that consists of a chemically-crosslinked polyacrylic acid (PAA), and its sensing mechanism relies not on conventional host-guest chemistry using tailored Ca2+-binding sites but on polymer-chain dynamics triggered by Ca2+. We show that ordinary PAA, when given pendants of tetraphenylethene (TPE), an aggregation-induced emission (AIE) luminogen33342+. This series of polymers  in the Ca2+ concentration in a flowing analyte containing ~1 mM Ca2+.Here we report a conceptually new fluorescent Ca2+-sensing receptor42+ concentration in extracellular regions and sends a signal to intracellular regions (362+-binding amino acid sequences42+ binding. This holds true for the extracellular Ca2+-sensing receptor (CAS) in plants2+-binding proteins382+ and clustering carboxylic acid domains in CaSR and CAS, we found an interesting analogy with a work of Flory2+ (25–50 mM). Subsequent reports, including those of Ikegami et al.et al.422+ causes the single-chain aggregation of PAA in water, for which [CO2−–Ca2+–−O2C]-type ion binding is responsible+, K+, Mg2+ and Ca2+), Ca2+ most effectively triggers such conformational change of the PAA chain404142As a clue for the design of the new sensor, we took notice of CaSR, a natural extracellular Ca regions . Unlike x with an expectation that the conformational change of PAA chain between aggregation and expansion upon binding and release of Ca2+, respectively, might be translated into the fluorescence property of the TPE pendants  of TPE pendants were synthesized by a two-step procedure involving the free-radical copolymerization of TPE-appended acrylate 1 and t-butyl acrylate 2 with the corresponding feed ratio (1/2 = 1/99–5/95) and the subsequent removal of t-butyl groups from the resulting copolymers using trifluoroacetic acid (x was unambiguously characterized by nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy (Mn) of PAA-TPEx to be approximately 20 kDa (Random copolymers PAA-TPEtroscopy . By meanx (10 mg/L) in a buffer solution ([4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)] = 70 mM, pH = 7.4) scarcely fluoresces, it becomes fluorescent upon addition of CaCl2. For example, the fluorescence intensity of PAA-TPE0.02 increased monotonically as the Ca2+ concentration was increased from 0.01 to 10 mM (2+ titration curves (x = 0.01–0.05) , a strong chelator for Ca2+ (Kd = ca. 10–10 M), was added to a buffer solution containing, e.g., PAA-TPE0.02 (10 mg/L) and Ca2+ (30 mM), the fluorescence was mostly quenched  experiments confirmed that the increase in the fluorescence intensity of PAA-TPE0.02 is due to single-chain aggregation4041422+ concentration was increased, the fluorescence intensity as well as the particle size  of PAA-TPE0.02 increased , the particle size of PAA-TPE0.02 increased . As shown in x). The apparent Kd decreased as the TPE content increased, suggesting that the hydrophobic nature of TPE promotes the Ca2+-triggered single-chain aggregation of PAA-TPEx. Importantly, because the relationship between the logarithm of the apparent Kd and the TPE content is approximately linear (Kd of PAA-TPEx can be continuously tuned in the range between 0.43 and 2.8 mM by simply varying the TPE content (x). This feature is beneficial for detecting a change in the Ca2+ concentration against various background concentrations of ions and provides an interesting contrast to typical molecular indicators such as the Fura seriesKd values of which are controlled by the electronic properties of the substituents on the BAPTA skeleton.Fitting the Caon curve  using Hix and in turn the enhancement of fluorescence intensity occurs very selectively for Ca2+. Without Ca2+, PAA-TPEx is weakly fluorescent in the presence of high concentrations of major ions in the body, i.e., Na+ (145 mM), K+ (5 mM) and Mg2+ (2 mM), as well as a physiological concentration (50 μM) of trace ions, i.e., Fe2+, Cu2+, Zn2+, Al3+, Sr2+ and Ba2+  containing PAA-TPE0.02 (10 mg/L), Na+ (145 mM), K+ (5 mM), Mg2+ (2 mM), glucose (14 mM) and glutamine (5 mM). Upon addition of CaCl2, the fluorescence of the buffer solution of PAA-TPE0.02 intensified (x can recognize Ca2+ selectively in the presence of such a high concentration of Mg2+ (Kd and the dynamic range (ratio of the maximum to the minimum fluorescence intensity) were determined to be 9.2 mM and 25, respectively. The difference of the apparent Kd for Ca2+ in the presence (9.2 mM) and absence (1.8 mM) of the biologically relevant ions and sugar likely arises from competing interactions of the carboxyl group of PAA with other metal ions and/or the polar functionalities of glucose and amino acid. This trend is generally observed for existing Ca2+ indicators2+ titration experiments under different pH  and temperature (25–40 °C) conditions, we confirmed that such pH and temperature changes exert little influence on the Ca2+ sensing property of PAA-TPE0.02  and acrylic acid 3 (95 mol%) were copolymerized in the presence of tetraethyleneglycol diacrylate 4 (3 mol%) as a crosslinker (0.02) was insoluble but swelled in aqueous media and could be readily processed into various shapes and sizes such as large-area flexible sheets and micro particles (2 (30 mM) was placed on a large-area gel sheet, blue fluorescence emerged  resulted in a less-swollen gel that scarcely responded to Ca2+ , indicating that the physical crosslinking due to polymer-chain entanglement is more important than the chemical crosslinking in determining the mobility of the polymer chain2+-sensing properties of the gel.The mM-order articles . When a  emerged , clearlysslinker  so that sslinker . The besnditions , entry 91 and acrylic acid 3 were copolymerized with varying molar ratios (1/3 = 1/96–5/92) in the presence of tetraethyleneglycol diacrylate 4 (3 mol%). The resultant materials  were all swollen in aqueous media and capable of sensing Ca2+ selectively . Hence, high-level loading of the TPE pendant (x = 0.05) would result in a decrease in the degree of freedom of the polymer chains to decrease the dynamic range. In fact, less-swollen g-PAA-TPE0.05 exhibited the smallest dynamic range among g-PAA-TPEx , ectively . Table 1eriments . Importag ratios . The TPEx could be used in various sizes and shapes . At BSA concentrations below 1.0 g/L, the fluorescence intensity of the gel monotonically increased, and then gradually decreased, mostly recovering its initial value at a BSA concentration of 20 g/L  containing 1.1 or 0.1 mM Ca2+. When these solutions were alternately flowed through the microfluidic channel with the gel, reversible changes in fluorescence intensity were observed in response to changes in the Ca2+ concentration , when an aggregation-induced emission luminogen is attached to its main chain, provides a state-of-the-art solid-state fluorescent Cage scale , has higge scale , and cant-butyl acrylate (2), acrylic acid (3), tetraethylene glycol diacrylate (4), 1,4-butanediol diacrylate (7) and 1,10-decanediol diacrylate (8) were purified by passage through Al2O3 column to remove polymerization inhibitors. Azobisisobutyronitrile (AIBN) and N,N′-methylenebis-acrylamide (6) were purified by recrystallization from methanol. 4-phenol (5) was prepared according to the reported procedureUnless otherwise noted, all the commercial reagents were used as received. Prior to use, See .3 (5 mL) solution of acryloyl chloride  was added dropwise at 0 °C to a CHCl3 (30 mL) solution of a mixture of 4-phenol  and triethylamine . The resulting mixture was stirred at 25 °C for 3 h, poured into a saturated aqueous solution of NaHCO3, and then extracted with CHCl3. A combined organic extract was washed with water and brine, dried over anhydrous MgSO4, and then evaporated to dryness under a reduced pressure. The residue was subjected to column chromatography  to allow the isolation of 1 as white solid (756 mg) in 72% yield: 1H NMR  δ (ppm) 7.01–7.11 , 6.89 , 6.56 , 6.27 , 5.99 . 13C NMR  δ (ppm) 164.3, 149.0, 143.7, 143.6, 143.5, 141.4, 141.3, 140.0, 132.4, 132.3, 131.4, 131.3, 128.1, 127.9, 127.8, 127.7, 126.6, 126.5, 120.7. FT-IR (KBr) ν (cm−1) 3076, 3054, 3024, 1756, 1677, 1599, 1502, 1443, 1356, 1200, 1166, 1140, 1017, 763, 748, 699, 613, 572, 498. HRMS (FAB): calcd. for C29H22O2 [M]+m/z = 402.1620; found: m/z = 402.1617.A CHCl1 , t-butyl acrylate  and AIBN  was degassed by freeze-pump-thaw cycles (three times) and purged with argon. The mixture was stirred at 60 °C for 12 h, allowed to cool to 25 °C, and then evaporated to dryness under a reduced pressure. The residue was freeze-dried from toluene to afford t-Bu-PAA-TPE0.05 quantitatively as white solid (167 mg): 1H NMR  δ (ppm) 6.79–7.11 (br), 2.05–2.39 (br), 1.71–1.86 (br), 1.20–1.63 (br). FT-IR (KBr) ν (cm–1) 2979, 2935, 1731, 1481, 1457, 1393, 1368, 1257, 1149, 1034, 909, 846, 751, 701, 471, 430. Using a procedure similar to that for t-Bu-PAA-TPE0.05, t-Bu-PAA-TPE0.01–0.04 were obtained quantitatively from monomer 1, t-butyl acrylate (2) and AIBN with the corresponding monomer feed ratios. The values of Mn and PDI of t-Bu-PAA-TPEx, evaluated by GPC analysis, are summarized in Typically, a dimethylformamide (DMF) solution (1.53 mL) of a mixture of monomer μL) was added to t-Bu-PAA-TPE0.05 . The mixture was stirred at 25 °C for 12 h and evaporated to dryness under a reduced pressure. The residual volatile compounds were azeotropically removed with methanol  to afford PAA-TPE0.05 quantitatively as white solid (9.8 mg): 1H NMR  δ (ppm) 6.79–7.21 (br), 2.28–2.65 (br), 1.40–2.22 (br). FT-IR (KBr) ν (cm−1) 2961, 2361, 1716, 1503, 1454, 1417, 1249, 1168, 802, 701, 614, 503, 414. Using a procedure similar to that for PAA-TPE0.05, PAA-TPE0.01–0.04 were obtained quantitatively from trifluoroacetic acid and the corresponding precursors (t-Bu-PAA-TPE0.01–0.04). The composition ratios of PAA-TPE0.01–0.04, evaluated by 1H NMR spectroscopy, are summarized in Typically, a trifluoroacetic acid , acrylic acid , tetraethylene glycol diacrylate  and AIBN  was degassed by freeze-pump-thaw cycles (three times) and purged with argon. The mixture was allowed to stand at 60 °C for 12 h and then cool to 25 °C. The resultant gelatinous material was subjected to Soxhlet extraction with a mixture of methanol/acetone (1/1 v/v) for 24 h, dried at 80 °C under a reduced pressure for 48 h, affording g-PAA-TPE0.05 as white solid (46 mg) in 48% yield. Using a procedure similar to that for g-PAA-TPE0.05, g-PAA-TPE0.01–0.04 were obtained in ~50% yield from monomer 1, acrylic acid (3), tetraethylene glycol diacrylate (4) and AIBN with the corresponding monomer feed ratios. The feed ratios for the preparation of g-PAA-TPEx are summarized in x, other crosslinked polymers , corresponding crosslinker (6–8) and AIBN with the corresponding monomer feed ratios.Typically, a DMF (0.71 mL) solution of a mixture of monomer polymers  were obt2+ titration curves were obtained by measuring the fluorescence intensities (for PAA-TPEx) or quantum yields (for g-PAA-TPEx) under various Ca2+ concentrations. Because the number of effective Ca2+-binding sites in PAA-TPEx and g-PAA-TPEx cannot be determined, a general stoichiometric analysis for determining the dissociation constant (Kd) is not applicable to these systems. Instead, we used the apparent Kd, which was obtained by fitting the Ca2+ titration curves with the following Hill’s http://www.R-project.org/).CaF: Fluorescence intensity (for PAA-TPEx) or quantum yield (for g-PAA-TPEx)Fmax: Maximum fluorescence intensity (for PAA-TPEx) or quantum yield (for g-PAA-TPEx)Fmin: Minimum fluorescence intensity (for PAA-TPEx) or quantum yield (for g-PAA-TPEx)Kd: Apparent dissociation constantn: Apparent Hill coefficientFor swelling the gel, a sliced sample was immersed in a HEPES buffer solution  at 25 °C for 30 minutes. Swelling ratios were evaluated from the following Wdry: The weight of dried g-PAA-TPExWswollen: The weight of swollen g-PAA-TPExAll experimental protocols were approved by the Animal Care Committee of Nara Medical University according to the NIH (USA) guidelines and the Guidelines for Proper Conduct of Animal Experiments published by the Science Council of Japan. Experimental details are described in How to cite this article: Ishiwari, F. et al. Bioinspired design of a polymer gel sensor for the realization of extracellular Ca2+ imaging. Sci. Rep.6, 24275; doi: 10.1038/srep24275 (2016).


Homeostatic and adaptive control mechanisms are essential for keeping organisms structurally and functionally stable. Integral feedback is a control theoretic concept which has long been known to keep a controlled variable  The biological motivation of this work can be summarized as follows: How can homeostatic mechanisms possibly work when many or even most of the regulatory processes within a cell are based on oscillations? Versions of this question and how oscillatory processes participate in homeostatic and adaptive mechanisms have been repeatedly asked and discussed Organisms have developed defending homeostatic mechanisms in order to survive changing or stressful conditions by maintaining their internal physiologies at an approximately constant level predictive homeostasisallostasisrheostasishomeodynamicsBecause many biochemical processes are oscillatory integral controlA) precisely and robustly at a given set-point A is generated A, while the other is the manipulated or controller variable E. Each controller consists of the two species A and E and three fluxes, the inflow and outflow to and from E and an E -controlled compensatory flux (either inflow or outflow) of A, denoted A caused by perturbations in other uncontrolled inflows/outflows of A. By considering activating or inhibitory signaling events from A to E and vice versa, eight basic negative feedback configurations  and by assuming first-order kinetics with respect to A in the rate equation for E, the set-point of A is given by (see Eq. S1 in (Dependent on whether q. S1 in ) (4)wherWhen A. In motif 2  E inhibits the compensatory flux, while in motif 5  the compensatory flux is activated by E. A limit-cycle version of motif 6 will be used to discuss cytosolic Ca2+ oscillations in terms of a homeostatic mechanism.To illustrate the homeostatic response of the oscillatory controllers, we use, as mentioned above, conservative and limit-cycle versions of inflow controller motif 2 and outflow controller motif 5 as representative examples. These motifs have been chosen, because they represent different ways to achieve negative feedback and homeostasis of the controlled variable H-function) can be found and for which the values of H remain constant in time. Conservative oscillators show periodic motions characterized by that they in phase space do not occur in isolation (i.e. they are not limit cycles). For a given H-level h a periodic motion (a closed path in phase space) is surrounded by a continuum of near-by paths, obtained for neighboring values of hH-function using the following equations: harmonic oscillatorLotka-Volterra oscillatorE. Here we also extend Goodwin's results by showing that limit-cycle oscillations can be created based on motif 2, but still using a cooperativity of 1 with respect to the inhibiting species E (see below).A conservative system is a system for which an energy or Hamiltonian function  all removal of A should either occur by zero-order kinetics with respect to A, or, when the removal of A, the formation of A needs to be a first (or nth)-order autocatalytic reaction H depends on the system's kinetics. Details on how H is constructed for the various situations is given in The following two requirements are needed to get conservative oscillations for any motif from  i.e., , , therebyIn this way the average level of tion see , which ition see  is givened when  . BecauseAt high The scheme in When vels in   are compFor the harmonic oscillators (motifs 1 or 5) The conservative oscillatory controllers described above can be transformed into limit-cycle oscillators by including an additional intermediate, and, as long as integral control is present, homeostasis in agnitude . As alrereserved .Similarly, a limit-cycle homeostat of motif 5 can be created  by inclu values, , homeostituation , the frerbations .In this section we present for the first time biochemical models that can show robust (perturbation-independent) frequency control. There are several biological oscillators where the frequency/period is under homeostatic regulation. Probably the best known example is the temperature compensation of the circadian period, i.e. these rhythms show an approximately constant period length of about 24 h at different but constant temperatures We show two ways how robust frequency control can be achieved. One is due to the presence of quasi-harmonic kinetics, i.e. the system, although still being a limit-cycle oscillator, behaves more like a harmonic oscillator. On basis of experimental results, we believe that the p53-Mdm2 system falls into this category see  below. Ithms see  below.approximately the steady-state assumption, i.e., quasi-conservative, because these systems still have a limit-cycle, but behave also as a conservative system. An interesting case occurs when the system is quasi-harmonic, i.e. when motifs 1 or 5 are used. In this case the limit-cycle oscillations and the frequency can approximately be described by a harmonic oscillator, i.e., a single sine function. This is illustrated in We consider now the case when the intermediate that has been implemented to obtain limit-cycle behavior  levels are under homeostatic control to concentrations at about 100 nM while extracellular levels are in the order of 1 mM. High Ca2+ concentrations are also found in the endoplasmatic reticulum (ER) and in mitochondria (between 0.1–10 mM), which act as calcium stores. To keep cytosolic Ca2+ concentrations at such a low level Ca2+ is actively pumped out from the cytoplasm into the extracellular space and into organelles by means of various Ca2+ ATPases located in the plasma membrane (PMCA pumps) and in organelle membranes 2+ signaling 2+ levels show oscillations 2+ oscillations have been found to occur in many cell types and differ considerably in their shapes and time scales with peak levels up to one order of magnitude higher than resting levels. Similar to the behavior of stimulated (perturbed) oscillatory homeostats as for example shown in 2+ oscillations have been found to increase their frequency upon increased stimulation of cells 2+ oscillations Cytosolic calcium  2+ into the ER and keeping the Ca2+ load in the ER high. It should be mentioned that the cause of the Ca2+ entry across the plasma membrane into the cytosol is not fully understood and different views have been expressed how this can occur Although a variety of mathematical models have been suggested to describe Ca2+ concentration in the ER is considered to be constant and only the pumping of Ca2+ from the cytosol into the extracellular space is taken into account without an increased cooperativity (Hill-function) with respect to the Ca2+ concentration. 2+ and the homeostat's performance at different inflow rates 2+ concentrations and/or different activation levels of the cell. As observed experimentally 2+ concentration or with an increased stimulation of the cell. As shown by 2+ homeostasis is preserved at varying Ca2+ inflow rates. In the absence of oscillations the Ca2+ concentration is still kept at its homeostatic set-point  feedback regulations in circadian pacemakers www.gnuplot.info)/Matlab. To make notations simpler, concentrations of compounds are denoted by compound names without square brackets. All concentrations, time units, and rate constants are given in arbitrary units (au).Computations were performed by using Matlab/Simulink (mathwork.com) and the Fortran subroutine LSODE File S1, contains derivation of the set-point under oscillatory conditions, construction of the H-function in conservative systems, the harmonic approximation of the frequency in conservative controllers, quenching of quasi-harmonic oscillations, and an alternative example of  feedback leading to robust frequency control in a motif 2 based limit-cycle oscillator.(PDF)Click here for additional data file.


Chronic lymphocytic leukemia (CLL) is a malignancy arising from a monoclonal population of mature B cells . ChronicWhile the natural progression of CLL varies according to the genetic and pathophysiologic characteristics of the cancer cells, treatment has traditionally depended more on patient factors than tumor factors. Chemotherapy regimens including both alkylating agents and purine analogs have shown significant efficacy against this disease, despite their inherent toxicities . Since nAlthough significant gains have been realized through the incorporation of newer less toxic, targeted therapies, there still remains a subset of patients with limited treatment options. These patients include those with unmutated IGHV, 17p deletions, and relapsed or refractory disease who typically respond poorly to current standards of care. With the exception of the anti-CD20 therapies, few targeted mechanisms have been available until recently. Our growing knowledge of B-cell receptor (BCR) signaling, however, has paved the way for many new agents currently under investigation. One of these new therapies is the phosphatidylinositol 3-kinase delta (PI3Kä) inhibitor idelalisib (Zydelig).BCR signaling, both antigen-dependent and independent, is critical to CLL survival and results from increased expression of certain survival pathways . Many ofPI3K produces a lipid product, phosphatidylinositol-3,4,5-triphosphate (PIP3), which serves to activate Akt, also referred to as protein kinase B or PKB . The tumIdelalisib, previously known as GS-1101 and CAL-101, in combination with rituximab was investigated in a phase III, multicenter, randomized, double-blind, placebo-controlled trial in 220 CLL patients with relapsed disease . EligiblThe 24-week PFS was 93% and 46% for the idelalisib and placebo groups, respectively, which resulted in the trial being stopped early due to treatment efficacy. The median duration of PFS was not reached in the idelalisib group and 5.5 months in the placebo group. The median duration of idelalisib and placebo treatment was 3.8 and 2.9 months, respectively, though 81% of idelalisib patients were continuing treatment at study termination compared to 52% of patients receiving placebo. The most impressive finding was that idelalisib and rituximab treatment had similar efficacy regardless of the presence of 17p deletion or IGHV mutational status. A secondary endpoint of overall survival was 92% vs. 80% in favor of idelalisib.Idelalisib has also been investigated in a phase II, single-arm, multicenter, open-label study in 125 patients with relapsed indolent B-cell NHL refractory to both rituximab and an alkylating agent . IdelaliIdelalisib is available in 100- and 150-mg tablets. The recommended starting dose is 150 mg given orally twice daily without regard to meals. Tablets should be swallowed whole. Refer to Idelalisib is mainly metabolized by CYP3A enzymes with minor metabolism via UGT1A4. Avoid concomitant use with strong CYP3A inducers  and monitor closely for increased risk of toxicities if used with strong CYP3A inhibitors . Caution is advised when coadministering CYP3A substrates, as idelalisib is a strong CYP3A inhibitor and dose adjustments or changes in therapy may be required .In the phase II trial, the five most common adverse events reported were diarrhea, nausea, fatigue, cough, and pyrexia. Grade 3 or 4 diarrhea or colitis occurred in 16% of patients after a median duration of 6 months. Idelalisib-related diarrhea appears to be late-onset and may not have been fully elucidated in the available trials. Other grade 3 or 4 events included neutropenia, elevated serum aminotransferases, thrombocytopenia, anemia, and pneumonitis, occurring in 27%, 13%, 6%, 2%, and 2% of patients, respectively. Other adverse events that were seen in greater than 10% of patients included decreased appetite, dyspnea, abdominal pain, vomiting, upper respiratory tract infection, decreased weight, rash, asthenia, night sweats, pneumonia, peripheral edema, and headache .In the phase III trial, more than 90% of patients experienced at least one adverse event. The most common adverse events in the idelalisib group were pyrexia, fatigue, nausea, chills, and diarrhea. Grade 3 or 4 neutropenia, thrombocytopenia, anemia, elevations in aminotransferases, and diarrhea occurred in 34%, 10%, 5%, 5%, and 4% of patients, respectively .Adverse event profiles were fairly similar in both trials . High inSevere adverse events and toxicities, including fatal or serious hepatotoxicity, diarrhea, or colitis were seen in both studies. This has prompted the Food and Drug Administration (FDA) to issue a Risk Evaluation and Mitigation Strategy (REMS) for idelalisib to make sure that practitioners are aware of the following black box warnings: fatal and/or serious hepatotoxicity, severe diarrhea, colitis, pneumonitis, and intestinal perforation. Aminotransferase monitoring should occur concurrently with CBC monitoring, as described above .Of note, lymphocytosis has been observed with many agents targeting the BCR pathway, including idelalisib. This effect usually peaks around week 2 and resolves by week 12, as noted in the phase III trial. Lymphocytosis may be blunted, however, when idelalisib is used in combination with other B-cell–depleting agents such as rituximab .Idelalisib was granted accelerated approval on July 23, 2014, as monotherapy for the treatment of relapsed follicular B-cell NHL and SLL . On the Idelalisib may be the first of several PI3K inhibitors to come. The pharmaceutical industry is actively pursuing PI3K inhibitors for many oncologic indications, including many solid tumor types, in light of the various PI3K isoforms and their roles in cell survival and development . AMG-319


More and more research groups have found that H2S mediates diverse cellular biological functions related to regulating intracellular calcium concentration. These groups have demonstrated the reciprocal interaction between H2S and calcium ion channels and transporters, such as L-type calcium channels (LTCC), T-type calcium channels (TTCC), sodium/calcium exchangers (NCX), transient receptor potential (TRP) channels, β-adrenergic receptors, and N-methyl-D-aspartate receptors (NMDAR) in different cells. However, the understanding of the molecular targets and mechanisms is incomplete. Recently, some research groups demonstrated that H2S modulates the activity of calcium ion channels through protein S-sulfhydration and polysulfide reactions. In this review, we elucidate that H2S controls intracellular calcium homeostasis and the underlying mechanisms.A growing amount of evidence has suggested that hydrogen sulfide (H Administration of NaHS (100 μM) significantly attenuated the effects of only ISO and forskolin. Moreover, NaHS reversed the ISO-induced cAMP increase and forskolin-stimulated adenylyl cyclase activity. Thus, they postulated that H2S may negatively regulate β-AR function through inhibition of the cAMP/PKA pathway. In addition, some studies found that the plasma concentration of H2S in patients with coronary heart disease i elevation in ECs pretreated with KB-R 7943. In addition, the amplitude of the Ca2+ response was significantly lower in ECs activated by the H2S donor in the presence of KB-R 7943. Taken together, these findings hinted at NCX as a key mediator of NaHS-elicited Ca2+ inflow in rat aortic ECs. To further determine the effect of sulfide signaling on the NCX, several studies investigated NCX expression and function in HeLa cells. They observed increased levels of NCX1 mRNA, protein, and activity after 24 h of GYY4137 (morpholin-4-ium-4-methoxyphenyl(morpholino) phosphinodithioate, a slow releasing H2S donor) treatment. This increase was accompanied by elevated cAMP due to GYY4137 treatment, which was completely abolished when NCX1 was silenced. An increased cAMP level would point to upregulation of the β-adrenergic receptors. Thus, Cheng et al. investigated the relationship of β-adrenergic receptors with the NCX1 in the presence and/or absence of H2S and determined the physiological importance of this potential communication using GYY4137 [β1 and β3 (but not β2) adrenergic receptors, suggesting that sulfide signaling played a role in regulating the NCX1 and β1 and β3 adrenergic receptors and their colocalization.The sodium calcium exchanger (NCX) is one of the key players in the regulation of intracellular calcium homeostasis. In a physiological condition, NCX, a nonselective cation channel, may induce the influx of 3 Naf 1 Ca2+ . Howeverthe cell . H2S may the NCX –46. To c GYY4137 . Indeed,2S and neuronal excitation induce calcium ion influx in astrocytes, and the interaction between neurons and astrocytes regulates synaptic activity [2S in the urinary bladder and sensory neurons [2S on transient receptor potential (TRP) channels are not completely clear, Kimura et al. demonstrated that polysulfides of H2S-derived signaling molecules stimulated TRP channels in the brain [2S induced Ca2+ influx in astrocytes through generating polysulfides of TRP. They administered sodium polysulfides, Na2S3, in their experiments, which induced Ca2+ influx in a concentration-dependent manner. They also confirmed that this astrocyte response to H2S was suppressed by the TRP channel blockers La3+ and Gd3+. To further reveal the mechanism for Na2S3-induced TRP channel opening, the TRPA1 channel inhibitors HC-030031 and AP-18 and TRPA1 siRNA were used. Their data showed that, in the presence of the inhibitors or TRPA1 siRNA, Na2S3 could not induce Ca2+ influx through the TRPA1 channel. Liu et al. showed that H2S maintained mesenchymal stem cell function via regulation of Ca2+ channel sulfhydration [2+ influx with a limited contribution from intracellular Ca2+ storage. They also found that DTT, by reducing the disulfide bonds in proteins and increasing the number of residual sulfhydryl proteins, elevated NaHS-induced Ca2+ influx in BMMSCs. Diamide, by reducing the number of sulfhydryls and 2-sulfonatoe-methanethiosulfonate (MTSES), a nonpermeable reagent able to reduce free sulfhydryls only on the outer cytomembrane, could reduce NaHS-induced Ca2+ influx in BMMSCs. These results revealed that free sulfhydryls affect NaHS-induced Ca2+ influx. The above results suggested that polysulfides, as H2S-derived bioactive molecules, stimulate TRP channels, providing a new molecular mechanism for sulfide-induced signaling.A growing body evidence has shown that Hactivity –50. TRP  neurons . Althoughe brain . They suydration . They fo2S, as a neuromodulator, elevates the activity of N-methyl-D-aspartate (NMDA) receptors to facilitate the induction of hippocampal long-term potentiation (LTP), a synaptic model of memory formation [2S enhances the neuronal response to glutamate and induces Ca2+ waves in astrocytes [2+ and propagating the signal as Ca2+ waves that occur in glia, and neurons show Ca2+ oscillations and intracellular Ca2+ waves. Because astrocytes elicit intracellular Ca2+ waves by electrical stimulation and application of NMDA in mixed cultures of neurons and astrocytes, astrocytes have been suggested to respond directly to a neurotransmitter released from neurons excited by NMDA or electrical stimulation [2+ and Gd3+ block Ca2+ waves and inhibit Ca2+ channels; La2+ and Gd3+ may inhibit the exocytosis of glutamate or some factor from neurons when neurons are stimulated by NMDA. However, La2+ and Gd3+ block H2S-initiated waves in pure astrocyte culture, showing that Ca2+ is most likely involved in the propagation step. H2S released in response to neuronal excitation may activate Ca2+ channels to induce Ca2+ waves in astrocytes. H2S may therefore mediate signals between neurons and glia. H2S is released from neurons or glia by neuronal excitation and increases the intracellular concentration of Ca2+ by activating Ca2+ channels in astrocytes and to a lesser extent causes release from intracellular Ca2+ stores. An elevated intracellular Ca2+ triggers the induction of Ca2+ waves that propagate to the neighboring astrocytes [2S enhances the activity of NMDA receptors by reducing the cysteine disulfide bond in the hinge region of the ligand-binding domain of NMDA receptors, and polysulfides further enhance this activity by adding bound sulfane sulfur to the receptors. Polysulfides activate the TRPA1 channels in astrocytes to induce Ca2+ influx, which facilitates the release of the gliotransmitter D-serine to enhance the activity of NMDA receptors. By these integrated mechanisms, H2S along with polysulfides may facilitate the induction of LTP [N-Methyl-D-aspartate receptors (NMDARs) form glutamate-gated ion channels that are widely expressed in the central nervous system and are highly permeable to calcium ions, which are essential for regulating synaptogenesis, use-dependent synaptic remodeling, and long-term plastic changes in synaptic strength . H2S, asormation , 55. Nagtrocytes . Glial cmulation –59. La2+trocytes –63. H2S n of LTP .2S is associated with relevant biological processes, such as cardiac systolic function, sensory transduction, antiapoptotic function, and neuroprotection [2S regulating various calcium ion channels and transporters [2S, have been found to elevate the activity of TRP, TTCC, and NMDARs and to inhibit LTCC through the mechanism of sulfhydration. Furthermore, H2S could upregulate the activities of the NCX1 and β1 and β3 adrenergic receptors and their colocalization. Altered effects of H2S on calcium ion channels under different pathophysiological conditions are being investigated. Extensive research on the mechanisms of H2S modulation of calcium signaling will provide new insights into the physiological function of H2S.An increasing amount of evidence has clearly demonstrated that Hotection . These fsporters . Many st