The assembly of the Mn4O5Ca requires light and involves a sequential process called photoactivation. This process harnesses the charge-separation of the photochemical reaction center and the coordination environment provided by the amino acid side chains of the protein to oxidize and organize the incoming manganese ions to form the oxo-bridged metal cluster capable of H2O-oxidation. Although most aspects of this assembly process remain poorly understood, recent advances in the elucidation of the crystal structure of the fully assembled cyanobacterial PSII complex help in the interpretation of the rich history of experiments designed to understand this process. Moreover, recent insights on the structure and stability of the constituent ions of the Mn4CaO5 cluster may guide future experiments. Here we consider the literature and suggest possible models of assembly including one involving single Mn2+ oxidation site for all Mn but requiring ion relocation.Photosynthetic water oxidation is catalyzed by the Mn PCC 6803 (hereafter Synechocystis 6803) lacking PsbO  and then released after interacting with a putative SRP receptor  with a carboxyl-terminal extension  whose length and sequence vary among different organisms  even in intact systems  can spontaneously convert to C in the dark with a 100–150 mshalf-time, with a kinetic constant designated kR in the scheme in Figure 1∕2 ~1–2 s, accounted for the optimum in light intensity or, alternatively, flash frequency, utilized for the assembly process. Photoactivation using saturating single turnover flashes is optimal with flash spacing of ~1 s, which is enough time to allow the dark rearrangement to occur (kR), but short enough to minimize the decay of the intermediate(s). If, however, the flash interval is too long, the second flash is not in time to trap forward progress and the reactants decay  involves a protein conformational change required for the binding and subsequent photooxidation of the second Mn2+  , will not be ready to utilize the second quantum and would thus the LED pulse would be effectively similar to a single turnover flash distributed in time over the population of centers. Variations and refinements of the original two-quantum model have been advanced based upon alternative techniques for illumination and O2 detection during photoactivation  low quantum efficiency and (2) the requirement for optimal flash spacing. However, inspection of data involving dark grown Chlorella indicates that photoactivation occurs more quickly, although optimal flash spacing is still required. Clearly, the role of assembly factors needs to be further pursued. One might imagine in this case that a closely associated assembly factor, like PratA, provides Mn2+ ions at critical times during the formation of intermediates and thereby mitigates potential losses due to intermediate decay. Except for one instance where the Psb27 mutant was analyzed  , D1-Glu333 affect the affinity characteristics of the high affinity binding site, but none as decisively as mutations of D1-Asp170  experiments have recently provided the evidence to support this hypothesis that the high-affinity Mn2+ site is located at the position denoted by Mn(4) in the crystal structure and the first photooxidized Mn2+ bound to the apo-WOC is coordinated with axial ligands Asp170 and Glu333 in the D1 protein , Mn(2), and Mn(3)  presumptive the first photo-oxidized MnSynechocystis 6803 PSII core complexes. Conventional perpendicular-mode EPR in X-band is used to detect spin transitions in half integer spin systems which satisfy the selection rules ΔMs = ± 1. Accordingly, the first light induced Mn3+ species, due to the integer spin S = 2 of Mn3+, is an EPR-silent species for perpendicular polarization EPR spectroscopy at X-band frequencies. X-band parallel polarization EPR spectroscopy, however, can be used to investigate integral spin systems with S ≥ 1 where the spin transitions satisfy the selection rules ΔMs = ± 2 and higher. This latter technique is therefore well-suited to examine the coordination environment of this Mn3+ intermediate (high spin S = 2). A six-line signal with a hyperfine splitting of ~45 G that was only visible in parallel mode. This signal clearly arises from Mn3+ as it closely resembles that observed for Mn3+ in superoxide dismutase . The Mn3+ parallel mode EPR signal gives an axial zero-field splitting value of D ≈−2.5 cm−1 and a rhombic zero-field splitting value of |E| ≈ 0.269 cm−1. The negative D value for this d4 ion is indicative of either an octahedral Mn3+ geometry or a five-coordinate square-pyramidal Mn3+ geometry. In contrast to wild-type, a different parallel polarization EPR signal of Mn3+ ion without a resolved hyperfine structure was observed in Asp170His mutant, suggesting a modified coordination environment of Mn3+. In the case of Asp170Glu mutant, instead of a parallel mode Mn3+ signal, a perpendicular mode signal generated by Mn4+ ion was detectable  of Mn2+ bound in a low-symmetry environment . Thus, when Ca2+ is left out of the photoactivation medium, binding and photooxidation of many more Mn2+ to the apo-WOC-PSII protein occurs, but no O2 evolution activity is observable  of Mn4CaO5 cluster, thus without the availability of the mature C-terminus Ala344, Ca2+ cannot bind and the destructive photoligation of Mn2+ to inappropriate sites can proceed. Additionally, competition between Ca2+ and Mn2+ for each other's binding sites has been indicated by many studies ]↔[Mn3+(OH−)]. The EPR-visible high-pH form exhibits a strong pH effect (pH 6.5–9) on Mn3+ spectral parameters, including the rhombicity (δ) derived from center field position (geff), the 55Mn hyperfine coupling (AZ), and the signal intensity. A pH-induced protein conformational change was proposed to account for the observed significant changes in the symmetry of the ligand field at the Mn3+ site. On the other hand, the EPR-detectable Mn3+ induced in the presence of Ca2+, exhibits a greatly weakened pH dependence of its ligand-field symmetry with reduced variation of rhombicity δ and 55Mn hyperfine coupling AZin the pH range of 6.5–9.0. Moreover, the addition of Ca2+ moves both geff and AZ to a range of values observed at alkaline pH ≥ 9 without added Ca2+, indicating that Ca2+ binding exerts an influence on the coordination shell of Mn3+ species equivalent to the alkaline pH effect in the absence of Ca2+. Therefore, it was proposed that Ca2+ binding induces a second ionization of the bridging hydroxo ligand bound to Mn3+ resulting in the formation of a bridging oxide ion ([Mn3+(OH−)-Ca2+] ↔ [Mn3+(O2−)-Ca2+]). The proton ionization of the water ligand is postulated to be controlled by a nearby base B−, which serves as an immediate proton acceptor with a pKa that depends upon the occupancy of the Ca2+ effector site. Looking at the current crystal structure and assuming a similar, although almost certainly not identical, spatial configuration of the Ca and Mn, the inferred oxo bridge would join these ions with D1-Asp170, Glu333 and the carboxyl terminus in proximity of one another.The effect of Ca4CaO5 cluster  likely occurs with high quantum efficiency, yet the photooxidation of the second Mn2+ (C⇒D) occurring after the rearrangement occurs with low quantum efficiency. There are essentially two general alternative hypotheses accounting for the low quantum yield of C⇒D that depend, in part, on the nature of the rearrangement. First, the rearrangement after A⇒B forms a binding site for the second Mn2+ that is not optimal for electron transfer to 2+ at the new site. Please note that the charge recombination may actually be the ‘rearrangement’ and could be important for removing mis-assembled clusters  and can exchange to another site. The EPR observations consistent with oxo bridge formation Ca2+ ion accompanying the photooxidation of Mn2+ ions at the high affinity site suggest a templating function for Ca2+ during assembly. Based upon the crystal structure, the Ca2+ would be captured between D1-Asp170 and the carboxyterminal carboxylate of the D1 protein. As noted, there is good evidence that Ca2+ binding induces a second ionization of the bridging hydroxo ligand bound to Mn3+ resulting in the formation of a bridging oxide ion ([Mn3+(OH−)-Ca2+] ↔ [Mn3+(O2−)-Ca2+])  bridge and facilitate the movement to the next site. Moreover, the water molecules coordinated Ca2+ may be provided as the substrate for additional μ-oxo bridge formation. In this way, Ca2+ functions in a manner similar to proposals for its role in catalytic water oxidation and substrate exchange though the positioning of coordinated waters .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chronic spontaneous urticaria is an itching skin disease characterised by wheals, angioedema, or both present for more than six weeks. Omalizumab is a humanized anti-IgE monoclonal antibody recently approved for treatment of chronic urticaria. Several randomised controlled trials have investigated the safety, tolerability, and efficacy of omalizumab for chronic urticaria. The safety of omalizumab in pregnancy is not known. We describe a female patient with chronic spontaneous urticaria who was treated with omalizumab continuously through two consecutive pregnancies with convincing results and no apparent toxicity. Urticaria is a severely itching skin disease characterised by wheals, angioedema, or both. It is divided into acute (present < 6 weeks) or chronic (present > 6 weeks) types . ChronicInternational guidelines recommend nonsedating antihistamines once daily as first-line therapy of CSU or CINDU; if no effect is observed it is recommended to increase the dose of antihistamine up to fourfold. If still refractory, third-line options include add-on therapy of omalizumab, ciclosporin, or montelukast .Omalizumab is a humanized anti-IgE monoclonal antibody recently approved for treatment of chronic urticaria , 2. SeveAlthough omalizumab apparently is safe and tolerable, based on the observations from clinical trials, data on long-term safety are still lacking. Notably, the safety and tolerability of omalizumab used in chronic urticaria during pregnancy have not been investigated, one exception being a woman from Germany, in whom no side effects or foetotoxicity was observed . We descThe patient was a 32-year-old woman with severe CSU with angioedema, atopic dermatitis, and asthma that was well controlled with terbutaline, who in August 2009 was referred to our department. At time of referral she also used the antidepressant escitalopram but did not require treatment for atopic dermatitis. The patient suffered from urticarial rashes primarily on the lower back, neck, dorsal side of hands, and the legs. She experienced symptoms of urticaria on a daily basis including particularly troublesome symptoms during night time that interfered with sleeping. A chest X-ray, urine and throat cultures, and routine blood tests including hepatitis B and C, tuberculosis, and helicobacter pylori were normal. Before treatment serum total IgE was 159 KIU/L (slightly elevated), whereas blood eosinophils were normal. She screened negative for antinuclear antibodies and a serum-induced basophil histamine release test was normal. For three months she was treated with high-dose nonsedating antihistamines (cetirizine 10 mg) up to three times daily but without effect. Hereafter, azathioprine 100 mg daily was added, and, after ten days she experienced a severe episode of facial angioedema that was treated with prednisolone for four days. She was then treated with an increased dose of 125 mg azathioprine in combination with nonsedating antihistamines four times daily due to the severe urticaria activity which could not be controlled alone on antihistamines.After six months without significant symptomatic relief, she was switched to the TNF inhibitor adalimumab (40 mg s.c. once every two weeks) and an improvement was observed; however hives were still emerging at the end of the dosing intervals. In August 2010, the patient was therefore switched to omalizumab 150 mg s.c. every two weeks and she then experienced a rapid (within the first 24 hours) and complete resolution of symptoms without any side effects or injection site reactions of the treatment. After six months' treatment she had an unusual asthmatic episode three days after receiving omalizumab, which was treated with 37.5 mg prednisolone for 10 days. It was not possible to clarify the trigger. In May 2011, omalizumab was paused to see whether the disease was permanently in remission, but already in June 2011 omalizumab was resumed due to symptoms of urticaria. In August 2011 she experienced a short-term exacerbation of urticaria while she was treated in hospital for a peritonsillar abscess.While still treated with omalizumab she became pregnant in November 2011, and, after having been thoroughly informed about the potential risks, she decided to continue treatment with omalizumab throughout pregnancy. In July 2012, she gave birth to a healthy girl with a birth weight of 4030 g and no congenital abnormalities. Due to prolonged pregnancy, labour was induced in week 42 of gestation. However, the child was delivered by acute Caesarean section due to slow progression of labour and failed vacuum extraction. The baby was not breastfed. Three years and two months after delivery the child is still healthy with no developmental abnormalities.After delivery the patient continued with her regular dose of omalizumab until she became pregnant again. As she was now experiencing complete remission of urticaria, the dose of omalizumab was changed to 300 mg s.c. once monthly. Again, no complications or side effects were observed during her second pregnancy and in September 2014 she delivered, by elective Caesarean section after week 39 of gestation, another healthy girl with a birth weight of 3500 g and no congenital abnormalities. The baby was not breastfed. Five months after delivery the child is still healthy with no developmental abnormalities .Clinical trials have shown that not only does omalizumab significantly reduce disease activity and symptoms of urticaria rapidly it is also well-tolerated and safe. Overall, the reported incidence of adverse effects is the same in patients treated with omalizumab compared to placebo-treated patients, although some studies , 7 did nWhile the majority of the patients in the clinical phase studies were women –7 pregnaOnly one previous case study  suggests that omalizumab is safe and effective during pregnancy . FurtherA limitation of our study is that the patient's urticaria activity was not followed prospectively with validated urticaria activity scores such as the UAS (Urticaria Activity Score), DLQI , or UCT (Urticaria Control Test) . These sOur report of successful and safe treatment of CSU with omalizumab during two consecutive pregnancies is important as many young women of childbearing potential suffer from urticaria. It is critical that an effective and safe treatment is provided for these patients even during pregnancy and lactation as urticaria has a substantial negative impact on quality of life.


Thus, it is not surprising that cardiac ICa is regulated by numerous pathways. This review will focus on changes in ICa that occur during the cardiac action potential (AP), with particular attention to Ca2+-dependent inactivation (CDI), Ca2+-dependent facilitation (CDF) and how calmodulin (CaM) and Ca2+-CaM dependent protein kinase (CaMKII) participate in the regulation of Ca2+ current during the cardiac AP. CDI depends on CaM pre-bound to the C-terminal of the L-type Ca2+ channel, such that Ca2+ influx and Ca2+ released from the sarcoplasmic reticulum bind to that CaM and cause CDI. In cardiac myocytes CDI normally pre-dominates over voltage-dependent inactivation. The decrease in ICa via CDI provides direct negative feedback on the overall Ca2+ influx during a single beat, when myocyte Ca2+ loading is high. CDF builds up over several beats, depends on CaMKII-dependent Ca2+ channel phosphorylation, and results in a staircase of increasing ICa peak, with progressively slower inactivation. CDF and CDI co-exist and in combination may fine-tune the ICa waveform during the cardiac AP. CDF may partially compensate for the tendency for Ca2+ channel availability to decrease at higher heart rates because of accumulating inactivation. CDF may also allow some reactivation of ICa during long duration cardiac APs, and contribute to early afterdepolarizations, a form of triggered arrhythmias.The cardiac voltage gated Ca That pore-forming α1 subunit also carries the intrinsic voltage-dependent gating properties  in most cardiac myocytes, and causes voltage-dependent activation of ICa. In pacemaker cells in the sino-atrial and atrio-ventricular node, it is ICa activation that is responsible for the rapid upstroke of the AP. ICa activation is a bit slower than INa activation, but starts early during the cardiac AP. The early repolarization phase of the AP (phase 1) can enhance ICa because of an increase in electrochemical driving force, i.e., membrane potential (Em) is further from the Ca2+ equilibrium potential  begin the processes of voltage- and Ca2+-dependent inactivation (VDI and CDI), which continues during the plateau phase of the AP (phase 2) causing a progressive decrease in ICa. As rapid and terminal AP repolarization ensue (phase 3) the LTCC undergoes de-activation, but then recovery from inactivation is both time and Em-dependent. Thus, for LTCC to recover full availability between beats, some time must elapse and that recovery time depends on Em .The rapid upstroke or phase 0 of the cardiac action potential (AP) is driven by NaCa amplitude and gating properties are influenced by myriad regulatory pathways, but here we will focus on the Ca2+-dependent mechanisms that shape the ICa occurring during the AP in ventricular myocytes. Hence, this review will describe how the Ca2+ sensing protein calmodulin (CaM) mediates CDI, and is involved in the activation of CaMKII, a serine/threonine-specific protein kinase which is a key mediator of ECC. Note that, although CaMKII activation can also be Ca2+-independent  of ICa, and how this process can eventually lead to Em or Ca2+ instabilities in ventricular myocytes.ICa is driven by VDI and CDI , local [Ca2+]i rises, causing Ca2+ to bind to CaM and induce inactivation. The details of the CDI process are not totally resolved, and may involve multiple regions of the channel, including the I-II loop that is thought to be key for VDI  would be associated with the IQ domain, and its N-lobe associated with the pre-IQ domain (between the IQ locus and the upstream EF-hand domain). Ca2+ binding to the N-lobe of CaM  would cause the N-lobe to shift and bind to part of the LTCC N-terminal domain , and thereby trigger N-lobe CDI. Then when Ca2+ also binds to the C-lobe of CaM  the C-lobe shifts its binding from the IQ domain to a position just upstream of the Pre-IQ region where the N-lobe had been bound. If Ca2+ binds only to the C-lobe  then the C-lobe does a similar sort of shift on its own, and mediates C-lobe CDI. For cardiac Cav1.2 channels, overall CDI and C-lobe-CDI are relatively similar, while N-lobe CDI alone was not apparent  increases from 17 to 37 ms when normal Ca2+ transients are abolished . Note that EGTA is a relatively slow buffer and cannot abolish very local [Ca2+] elevation around the mouth of the channel . In absence of extracellular Ca2+, LTCC are permeable to Ba2+, and this current (IBa) has been often studied to differentiate VDI and CDI , IBa inactivation is further slowed (t1/2 = 161 ms).Figure NS, mostly carried by Na+ and Cs+). INS inactivates only very slowly at this voltage at room temperature  even at more positive Em , reflecting the contribution of CDI. Note that at +50–60 mV little Ca2+ enters during ICa, and the extent of ICa and INS inactivation is similar. It is tempting to speculate that INS inactivation properties might provide pure VDI characteristics that are relevant for ICa. However, INS can actually inactivate faster than IBa at positive voltages, so we think that using INS to assess VDI characteristics for ICa is likely to be invalid  regulation. Inhibition of ICa inactivation induces AP prolongation, and has pro-arrhythmic consequences (see section “Arrhythmogenic consequences of CaMKII-dependent ICa effects”). For example, impaired VDI has been observed in Timothy syndrome  in adult guinea-pig cardiomyocytes also prevents CDI and causes dramatic AP prolongation  was thought to be responsible for the increased APD seen in HF. Only in the late 1990s the pivotal role of CDI became clear, when it was first proposed in a theoretical study in dog . Conversely, when myocyte Ca2+ is low and SR Ca2+release is small, there is less CDI and enhanced Ca2+ entry that increases intracellular Ca2+ content .APD regulation is fundamental to control the CaCa during the AP is significantly different compared to that seen during a square voltage pulse i on unitary ICa have been reported  and CDI are responsible for the observed CDF , the active CaMKII must be highly localized near the channels  or abolish (Ile to Glu) CDF  in Ser-1512 and Ser-1570 , and abolished by the double mutation S1512A/S1570A  phosphorylation sites, and confirmed that Cav1.2 channel is modulated by CaMKII-dependent phosphorylation in the murine heart.More recent studies in heterologous cells indicate that CaMKII may directly bind and phosphorylate the α1C subunit. In oocytes CaMKII could phosphorylate the α1C subunit  because of the transient inward current carried by the Na+/Ca2+ exchanger (in the Ca2+ extrusion mode). In a theoretical study  is prevented by CaMKII inhibition (with KN-93 or AC3-I) , and ICa could be reduced back to control levels by blocking CaMKII with KN-93 or AIP  genetically targeted to the SR, consistent with the notion that CaMKII involved in ICa facilitation being localized at junctions between the SR and sarcolemma. Interestingly, Xu et al. . We recently extended their work, by studying the synergy of Na+ handling with Ca2+ and CaMKII signaling, since CaMKII hyperactivity in HF has also been associated with late INa and intracellular [Na+] ([Na+]i) overload , which is what happens in HF . However, when this system is chronically on in pathological states it may contribute to inappropriate Ca2+ loading of the myocytes, and contribute to worsening pathology via poor diastolic function or arrhythmias triggered by EADs or DADs, altered ICa restitution or cardiac alternans. The detailed molecular mechanisms remain to be fully resolved, but work over the past 10–20 years has paved the way for further clarification in the near future.CaMKII has numerous targets in cardiac myocytes, and we must assume that under normal physiological conditions this orchestrates a response that is acutely adaptive. However, when CaMKII becomes chronically activated in disease, by autophosphorylation and oxidation (Anderson et al., Donald M. Bers received a research grant from Gilead Sciences in May 2013. Gilead Sciences was in no way involved in the design, funding, execution, or interpretation of this study. Stefano Morotti has nothing to disclose.


Our previous studies in guinea pig VHCs II indicated that acetylcholine (ACh) evoked the BK current by triggering the influx of Ca2+ ions through l-type Ca2+ channels, which was mediated by M2 muscarinic ACh receptor (mAChRs). Aminoglycoside antibiotics, such as gentamicin (GM), are known to have vestibulotoxicity, including damaging effects on the efferent nerve endings on VHCs II. This study used the whole-cell patch clamp technique to determine whether GM affects the vestibular efferent system at postsynaptic M2-mAChRs or the membrane ion channels. We found that GM could block the ACh-induced BK current and that inhibition was reversible, voltage-independent, and dose-dependent with an IC50 value of 36.3 ± 7.8 μM. Increasing the ACh concentration had little influence on GM blocking effect, but increasing the extracellular Ca2+ concentration ([Ca2+]o) could antagonize it. Moreover, 50 μM GM potently blocked Ca2+ currents activated by (−)-Bay-K8644, but did not block BK currents induced by NS1619. These observations indicate that GM most likely blocks the M2 mAChR-mediated response by competing with Ca2+ at the l-type calcium channel. These results provide insights into the vestibulotoxicity of aminoglycoside antibiotics on mammalian VHCs II.Type II vestibular hair cells (VHCs II) contain big-conductance Ca However, widespread use of aminoglycosides has been restricted because of the incidence of serious side effects, such as nephrotoxicity, ototoxicity and muscle paralysis. Moreover, intratympanic application of gentamicin (GM), an ototoxic aminoglycoside, could be efficacious for treating vertigo in Meniere’s disease because it is more toxic to vestibular hair cells (VHCs) than cochlea hair cells –3. Most nAChRs) o) and then observed the blocking effect.Since GM competed with Ca2+]o from 2 to 4 mM, and that the current amplitude did not increase at concentrations higher than 4 mM [2+]o solutions. As shown in n = 6) to 40.3% ± 8.4%  upon increasing [Ca2+]o from 2 to 4 mM. These results showed that GM could block BK currents in both normal and higher [Ca2+]o solutions and the blocking effect was weaker in the elevated [Ca2+]o condition. These results indicate that Ca2+ may compete with GM to antagonize GM inhibitory effect.Our previous study showed that the ACh-induced BK current amplitude increased with the change of [Cahan 4 mM . Therefo2+ and can activate small-conductance Ca2+-dependent K+ channels in guinea pig VHCs II. Although our previous findings showed that the nAChRs were not involved in the BK currents recorded [2+ influx though nAChRs was greater with higher [Ca2+]o. As shown in 2+]o.  or 4 mM [Ca2+]o.  solutions. These data indicated that there was no Ca2+ influx through nAChRs even in high [Ca2+]o solution. The nAChR, which is not involved in BK currents, only affected the antagonism of elevated calcium concentrations by increasing Ca2+ influx. Therefore, we could rule out the involvement of nAChRs in the antagonism of GM blocking effect under elevated [Ca2+]o conditions.It is known that nAChRs have high permeability to Carecorded , it was 2+ influx, which was not mediated by nAChRs. Our previous studies have reported that ACh evoked BK currents by triggering Ca2+ influx through l-type Ca2+ channels o could antagonize GM blocking effect, which indicated that GM may block the BK current by impairing calcium influx. It has been reported that GM could impair the calcium influx from the calcium channels [2+ concentration alleviated GM blocking effect through both or only one of them. Our previous findings showed the nAChRs were not involved in the activation of BK channels and BK currents evoked by ACh was insensitive to strychnine [2+]o solution. Recently, we verified that nAChRs were deactivated in collagenase IA-isolated VHCs II [2+]o on the GM blocking effect was not due to nAChRs, but rather the l-type Ca2+ channel. To obtain more direct evidence of GM competing with Ca2+, we recorded the Ca2+ current evoked by (−)-Bay-K8644 and observed the direct effect of GM on it. The results showed that both 50 and 300 μM of GM potently inhibited the Ca2+ currents, indicating that GM could block l-type Ca2+ channels and decrease the influx of Ca2+. GM slightly blocked NS1619-activated BK currents at a high concentration of 300 μM, so the BK channel might be the blocking target of GM. Some studies have also shown that aminoglycosides could directly block the K+ channel o. Therefore, the direct blocking of BK channels might be a very small explanation, but is not the major mechanism.The current findings demonstrated that increasing [Cachannels ,31,32 anchannels . In guinrychnine . The pre VHCs II . Therefo channel –37; howe2+ influx through l-type calcium channels, but not the release of intracellular Ca2+ stores [2+ at the l-type calcium channel, which impaired the calcium influx to diminish the BK current. This provided a good explanation for the absence of increased K+ efflux even after the activation of more M2 mAChRs. Martini et al. [2+-dependent K+ current by impairing the Ca2+ influx in semicircular canal hair cells of the frog.Our previous study demonstrated that the activation of ACh-induced BK current was mainly dependent on external Ca+ stores . Based oi et al.  have rep50 (36.3 μM) of GM to the ACh-induced BK current mediated by the M2 mAChR was higher than GM (5.5 μM) to ACh-evoked K+ currents in outer hair cells at the nAChR. This may be due to differences in cell type or cholinergic receptor type.Our results also showed that GM reversibly blocked the ACh-induced BK current in guinea pig VHCs II in the micromolar range. GM also blocked the ACh-induced small-conductance calcium-dependent potassium current in guinea pig outer hair cells . However4.4.1.The Institutional Animal Care and Use Committee of Tong-ji Medical College approved this animal experiment on 1 June 2010 (IACUC Number: 289).4.2.2+ and Mg2+-free balanced salt solution ). Next, the low calcium solution was replaced with normal external solution containing 2 mM CaCl2 to stop the enzymatic action. VHCs were isolated by mechanical dissociation and placed on the bottom of the experimental chamber, which was coated with rat collagen. We identified VHCs II by the cylindrical shape and absence of a distinct neck region [Collagenase type IA-dissociated vestibular hair cells were isolated as previously described –29. Firsk region .4.3.Cm, n = 6) and the series resistance  was compensated by up to 80%. The drug was administered to a patched cell at a holding potential of −50 mV.The ACh-induced BK currents were recorded in the whole-cell configuration, using an Axon-200B patch clamp amplifier . Patch electrodes were fabricated from thick-walked borosilicate glass capillaries, using a Model P-97 electrode puller . Electroderesistances were maintained between 3 and 6 MΩ when filled with the internal solution, as described below. Records were low-pass filtered at 5 kHz with a four-pole Bessel filter. After gigaseal formation onto the basolateral membrane of VHCs and membrane disruption, the membrane capacitance was 4.8 ± 1.7 pF on average . The components of the internal solution were as follows: 150 mM KCl, 2 mM MgCl2, 3 mM Na2ATP, 0.1 mM EDTA and 10 mM HEPES (pH 7.2). The KCl was replaced by CsCl when the calcium current was recorded.The components of the external solution were as follows: 150 mM NaCl, 5 mM KCl, 2 mM CaCl4.4.nAChR inhibitor), and iberiotoxin  were directly dissolved in the external solution. NS1619 (a BK channel activator) was formulated as a 10 mM stock solution in DMSO . (−)-Bay-K8644  and nifedipine  were formulated as 10 mM stock solutions in DMSO and diluted for use. The test solutions were dissolved daily before use and applied to cells by a gravity-delivered linear barrel microperfusion system as previously described [All drugs were purchased from Sigma . ACh, gentamicin (GM), methoctramine (an M2 selective AChR antagonist), strychnine  to compare the means among more than two groups. Differences were considered to be significant if p < 0.05; all the differences listed were statistically significant, unless stated otherwise.Data were analyzed and plotted by using the pCLAMP8.1 Clampfit 8.1 software  and SigmaPlot 9.0 . Results were presented as the mean ± SD. Statistical significance was determined using the Student’s 5.2+ at the l-type calcium channel, which results in the decrease of Ca2+ influx and the subsequent reduction of the BK current. Thus, the effect of GM on nAChRs in guinea pig VHCs II should be further investigated.In conclusion, our findings indicate that acute GM application could block the ACh-induced BK current in guinea pig VHCs II by competing with Ca


Phase 1 is characterized by a large increase of the intracellular Ca2+ concentration ([Ca2+]i) with a minimal reduction of the free luminal SR [Ca2+] ([Ca2+]FSR). Importantly, active SR Ca2+ ATPases (SERCA pumps) are necessary for phase 1 to occur. This situation cannot be explained by the standard kinetics that involves a fixed amount of luminal Ca2+ binding sites. A new mathematical model was developed that assumes an increasing SR Ca2+ buffering capacity in response to an increase of the luminal SR [Ca2+] that is called Kinetics-on-Demand (KonD) model. This approach can explain both phase 1 and the refractory period associated with a recovered [Ca2+]FSR. Additionally, our data suggest that active SERCA pumps are a requisite for KonD to be functional; otherwise luminal SR Ca2+ binding proteins switch to standard kinetics. The importance of KonD Ca2+ binding properties is twofold: a more efficient Ca2+ release process and that [Ca2+]FSR and Ca2+-bound to SR proteins ([Ca2+]BSR) can be regulated separately allowing for Ca2+ release to occur  without an initial reduction of the [Ca2+]FSR.The process of Ca These membrane organelles are distributed throughout the cell and are endowed with three main elements 1) SERCA pumps, 2) different types of Ca2+ release channels , and 3) luminal Ca2+-binding proteins . All these different types of proteins produce and modulate Ca2+ release events that can be either localized in nature or traveling Ca2+ waves [The main internal Ca2+ stores via ATP hydrolysis [2+] ([Ca2+]FSR) and by different type of proteins FSR [2+ level [2+ release and eliminates [Ca2+]i gradients before any reduction of the ER Ca2+ store [2+ wave in heart cells [2+]FSR, reduces the histamine-induced potentiation of IP3-induced Ca2+ release [2+]i responses FSR . Additio2+ level  and acti2+ level ,7. In pa2+ store . More rert cells . In HeLa release . In smooesponses ,11. Morele cells . Interesoduction . Overexp2+ store . Collect2+ with low affinity and high capacity, for instance calsequestrin and calreticulin. Interestingly, the Ca2+ buffer capacity of calsequestrin increases with the degree of polymerization, from monomer to dimer and to polymer [2+ with minimal reductions in the [Ca2+]FSR [2+ with minimal modification of the [Ca2+]FSR FSR is observed [2+]FSR is similar for the smaller Ca2+ sparks than for the larger Ca2+ waves [2+]FSR cannot be correlated with the total amount of Ca2+ released by the SR so we have hypothesized that there is a concealed source of luminal Ca2+ for release channels [SR and ER also contain a large variety of proteins that bind Ca polymer . These cCa2+]FSR ,16–19 i.Ca2+]FSR . The preobserved . Additio2+ waves . All the2+]FSR and the amount of Ca2+-bound to luminal proteins ([Ca2+]BSR), to test this hypothesis a simple deterministic mathematical model was developed, in order to understand how release channels (particularly RyRs), luminal SR proteins and SERCA pumps work together to facilitate Ca2+ release in smooth muscle cells. This new model suggests that luminal SR Ca2+ binding proteins are working different to what it is commonly believed, since the number of apparent Ca2+ binding sites increases as a function of the free luminal SR [Ca2+], this situation we have called Kinetics on Demand (KonD), and requires active SERCA pumps to have an efficient Ca2+ release event.Since it is not feasible yet to look simultaneously at both [CaAll animal care and experimental procedures were performed in compliance with the Mexican Official Norm for the Use and Care of Laboratory Animals (NOM-062-ZOO-1999) and the protocol was approved by the local Ethics Committee on Animal Experimentation (CICUAL-Cinvestav) with the reference number 0306–06 and renewed with the reference number 0131–15. Animals were bred and housed in Cinvestav Animal facility with clean air and controlled both light and temperature. Food and water was given ad libitum. Precautions were implemented to minimize animal use and reduce pain and distress.2+]i as previously reported [2+]i and the free luminal SR Ca2+ level ([Ca2+]FSR) in these cells was carried out using fura-2 and Mag-Fluo-4, respectively; as previously described [2+ release was induced by application of 2 or 20 mM caffeine with a puffer pipette [Male albino guinea pigs of 400 to 490g weight  were killed by decapitation followed by exsanguination. Single smooth muscle cells were isolated from guinea pig urinary bladder and loaded with fura-2 to record [Caescribed . Ca2+ re2+]Ti and [Ca2+]TSR represent the total [Ca2+] in the respective compartments. Let J1 be the Ca2+ flux from the cytoplasm to outside the cell together with all cytoplasmic Ca2+ removal mechanisms (except SERCA pumps) that are involved in keeping a constant basal concentration of this ion, J2 be the Ca2+ flux from the SR to the cytoplasm via RyRs present in the SR membrane, and J3 be the Ca2+ flux from the cytoplasm to the SR via SERCA pumps located in the SR membrane. It follows from these considerations thatFor the purpose of the present model we have considered that smooth muscle cells consist of two compartments: the cytoplasm and the SR , and i is the free cytoplasmic [Ca2+], while 2+]i, and a is the first order kinetic constant for this flux. We have assumed that no membrane potential exists across the SR membrane [2 is driven by the gradient of free Ca2+ ions across this membrane:b is a constant parameter proportional to the maximum [Ca2+] flow through a single RyR, Po is the probability that a single RyR is open as a function of [Ca2+]i and of the caffeine concentration ([Caff]) used to induce Ca2+ release from SR store, [Ca2+]FSR is the concentration of free Ca2+ in the SR, and γ being the volume ratio between SR and cytoplasm. SR appears to have a complex fractal-like structure, hence its surface should scale with respect to the volume according to a power law nv is a parameter related to the SR fractal dimension , so it follows that:In the cytoplasm, CaBT represents the total amount of Ca2+ binding sites with a dissociation constant KD.Assuming infinitesimal small increments, Eq  can be s2+ bound to proteins in a cell. However, it is rather easy to determine the total amount of Ca2+ added to the cell ,and in t that Eq  by the f2+ buffering of smooth muscle and chromaffin cells appears to be constant [2+] as:Interestingly, the cytoplasmic Caconstant ,25. We h2+ ions bind this channel on its cytoplasmic side, a process regarded as Ca2+ induced Ca2+ release (CICR), which functions at low efficiency in smooth muscle cell /KF ≪ 1, the above expression can be rewritten as:kf = kF / KF. Finally, considering that Ca2+ needs to be bound to all four subunits in order for the channel to open, and that there is a certain amount of cooperativity among subunits, the RyR open probability can be written as  does not change, a situation that might reflect the ability of luminal Ca2+ buffering proteins to have a KonD type of Ca2+ binding. This observation is compatible with the assumption that new Ca2+ binding sites become apparent as the already available ones are bound to Ca2+. Since there is no mathematical framework to model this type of behavior, we decided to test this new paradigm by assuming that Eq , which is followed by a large reduction of [Ca2+]FSR and a plateau of the [Ca2+]i increase (phase 2). Additionally, it was found that rapid inhibition of SERCA pump reduces both the amplitude and the rate of rise of the caffeine-induced [Ca2+]i response [2+]i (upper trace) and the [Ca2+]FSR (middle trace) before and after the inhibition of SERCA pumps with thapsigargin. Application with a second puffer pipette of 10 μM thapsigargin for 5 sec was enough to inhibit completely the recovery of the [Ca2+]FSR after terminating the second application of caffeine  and the effect of rapid inhibition of SERCA pump with thapsigargin on the 20 mM caffeine-induced [Ca2+]i response. The mathematical model is based on a new concept named Kinetics-on-Demand (KonD), where Ca2+-binding sites of SR Ca2+-binding proteins are an increasing function of [Ca2+] . The KonD model was also able to reproduce the relation between the peak rate of rise and the rise time of the caffeine-induced [Ca2+]i responses  with a second puffer pipette for 5 seconds and 30 seconds before the application of caffeine resulted, as expected, in smaller  and slow2+]i and the reduction of the [Ca2+]FSR. We have used KonD model to determine which parameters need to be changed to reproduce the effect of thapsigargin. This exercise is shown in 2+]i responses to the application of 20 mM caffeine where indicated, while 2+]FSR and 2+] ([Ca2+]TSR) which corresponds to the sum of both [Ca2+]BSR and [Ca2+]FSR. The complete inhibition of SERCA pump by itself did not importantly alter (red line) the [Ca2+]i response to the application of 20 mM caffeine  and the changes in the free luminal SR [Ca2+] (lower traces) in response to the dual application of saturating concentrations of caffeine (middle trace). The parameters used here are exactly the same to those shown in 2+]i response. The same parameters were used for the SK model. Since caffeine released the same amount of Ca2+ from the SR for both models, the reduced amplitude of the [Ca2+]i response seen with the standard model was due to the competition for Ca2+ between the luminal SR Ca2+ binding proteins and the release channels, a situation that is not observed in the KonD model as demonstrated in 2+] slightly precedes the recorded reduction of the luminal SR [Ca2+]. On the other hand, KonD model shows an exaggerated phase 1. Interestingly, the KonD model shows a rapid recovery of the free luminal SR [Ca2+] that does not involve the recovery of the total SR [Ca2+]  increases as [Ca2+] is increased, i.e. Bmax = 22 in the range of 0 to 0.5 mM [Ca2+], and goes to 40 between 0.5 and 3 mM [Ca2+], Bmax increases to 65 for the range of 3 to 7 mM [Ca2+] and increases again to 79 from 7 to 20 mM [Ca2+] 2+ releasf the ER .2+ release and the transition from phase1 to phase 2. Phase 1 represents a condition of a very high Ca2+ buffer capacity since there are large changes in the [Ca2+]i with non-existent  or miniondition . Howevercapacity . Accordimentally ,16,30. Al [Ca2+] ,31,32. Ilylysine . Likewishis work . Interesof IP3Rs . Later oce of K+ . Interesh or low . These dl muscle .2+]FSR and a much slower recovery of the [Ca2+]TSR  conformational activation of release channels, 2) induction of Ca2+ release from the luminal proteins and the subsequent increase in the luminal [Ca2+] and 3) opening of release channels and Ca2+ moving from the SR to the cytoplasm  would trigger the opening of the release channel and allow Ca2+ movement to the cytoplasm  [2+ release in HeLa cells [2+ wave without any reduction in the caffeine-sensitive store [2+ release in the absence of an increased loading of SR Ca2+ store [2+ release for both RyRs and IP3Rs [2+]FSR i transient.Inhibition of SERCA pumps reduces CaR [Ca2+] . AdditioLa cells . Rapid ive store . The opp2+ store . SERCA pnd IP3Rs , this wa [2+]FSR ,11. Coll2+ buffering capacity with a positive curvature; however, although likely in the right direction might not be the right solution. This is because another characteristic of SR Ca2+ stores is that Ca2+ freely diffuses in the lumen, a condition that is not expected from the KonD condition. The most likely solution is that the trapping of Ca2+ by luminal SR Ca2+ binding proteins does not occur instantaneously because it depends on the conformational changes and aggregation of proteins, a situation that might take a longer time than the one required for free diffusion of Ca2+ in the lumen of SR, so KonD model should consider that the kinetics of dissociation of Ca2+ from luminal SR proteins is rather fast while the kinetics of association is rather slow.KonD condition is the simplest solution, we were able to visualize for an SR Ca2+ spark termination in heart cells has contemplated Ca2+ diffusion inside the SR  as in A but using KonD model and Eq ((A) Typical relationship for a 3 mM Causing Eq . (B) Ca2l and Eq  with val(TIF)Click here for additional data file.S3 Fig2+ bound to calsequestrin as a function of [Ca2+]; data replotted from 2+ binding sites (Bmax) is increasing as a function of the [Ca2+]. The inset shows the fitting for [Ca2+] in the range of 0–0.6mM. Park et al. have shown that blocking polymerization of calsequestrin inhibits this effect of increasing Bmax, so Bmax value stays around 22 for the whole range of [Ca2+] tested (triangle line) [max value higher than 70.Models of Cale line) . At leas(TIF)Click here for additional data file.S4 Fig2+]i response (blue line). Although there is now a good fitting of the [Ca2+]i responses induced by caffeine, the time course of recovery of the [Ca2+]FSR is much slower in the SK model than is recorded by the Mag-Fluo-4 indicator. This can be explained by considering that free and total Ca2+ go together in the SK model, while in the KonD model  was modified to fit the caffeine-induced [Casee Figs  and 7 th(TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


The effect of endogenous plant Ca2+ concentrations on tipburn was also studied in a doubled haploid (DH) population consisting of 100 individuals, but no correlation was found. We then examined the expression of 12 Ca2+ transporter genes that function in cytosolic Ca2+ homeostasis in both tipburn-susceptible and tipburn-resistant plants under normal and tipburn-inducing conditions. Expression patterns for most of these genes differed between the two types of plants. Salicylic acid (SA) accumulated in response to conditions of calcium deficiency in our study, and both total SA and SA β-glucoside (SAG) in tipburn-susceptible plants was ∼3-fold higher than it was in resistant plants following Ca2+ deficiency treatment. Also, the changes observed in SA levels correlated well with cell death patterns revealed by trypan blue staining. Therefore, we speculate that the cytoplasmic Ca2+ fluctuation-induced downstream signaling events, as well as SA signaling or other biological events, are involved in the plant defense response to tipburn in Chinese cabbage.By cultivating tipburn-susceptible plants in modified Hoagland’s medium containing of gradient exogenous calcium (CaThe online version of this article (doi:10.1007/s11105-015-0949-8) contains supplementary material, which is available to authorized users. Tipburn is one of the irreversible physiological disorders that causes large economic losses in the production of crops such as cabbage, Chinese cabbage, lettuce, cauliflower, strawberry, and tomato Nieuwhof . Typical2+) is a contributing factor , but it is the cytosolic Ca2+ that shows significant concentration changes (Ca2+ oscillations) and plays central roles in the host response to various stress signals  and CAX (Ca2+/H+ antiporters) proteins, and ER-localized ECA (P2A-type Ca2+-ATPases) proteins, which catalyze Ca2+ efflux to dampen cytoplasmic Ca2+ concentration changes , with the strong appearance of scattered macrolesions around the leaf, especially at the leaf margin. These growth defects can be suppressed by exogenous Ca2+ , can result in an enhanced resistance to calcium-limiting conditions and/or improved stress resistance under such conditions in Arabidopsis, tobacco, and tomato , which binds a 50-fold excess of Ca2+ and may facilitate Ca2+ transport across plasmodesmatal ER, causes a dwarf phenotype with an increased level of SA in transgenic Arabidopsis plants  is required for local and systemic disease resistance responses in higher plants, and SA-dependent cell death has been reported to correlate closely with Ca2+ deficiency and tipburn incidence, we firstly demonstrated the correlation between tipburn severity and plant exogenous, intrinsic Ca2+ levels, and SA concentrations in Chinese cabbage. In addition, the transcriptional changes of the Ca2+ transporter genes, and SA biosynthesis and response genes were also studied under different Ca2+ concentration or infection conditions. Our study will provide valuable information to understand the mechanism of tipburn incidence in Chinese cabbage.To further understand the relationship between Ca1 of the cross between the female parent “Orange Queen” (OQ) and the male parent “QinXiao No. 2” (QX). The inbred line OQ is highly susceptible to tipburn, while QX is highly resistant.A population consisting of 100 DH lines was derived by microspore culture from an F1BC4 individuals was obtained by four generations of backcrossing. The F1 was generated by crossing the tipburn-susceptible parent “Sheng Xiao” (SX) with the tipburn-resistant parent “DaBaiKou” (DBK), which was also used as the recurrent parent.Another population consisting of 285 F1BC4 populations and the parental inbreds were planted following a randomized complete block design with three replications in the experimental field at Beijing Vegetable Research Center  in the autumn of 2013. For the DH population, three replicates were conducted, with 10 plants per replicate (n = 30). A higher nitrate supply and less water compared to normal cultivation were applied to induce tipburn during the crop growing period. The symptoms and disease index (DI) of tipburn were observed when the plant heads reach acceptable commercial firmness.For field evaluation, the DH and Fw/v) agar medium supplemented with 3 % (w/v) sucrose and grown until the first two leaves had formed. The seedlings were transferred to a modified liquid medium which consisted of Hoagland’s medium at pH ∼6.0 containing four levels of Ca2+ . The Hoagland’s medium containing 0.57 and 5.7 mM CaCl2 were defined as Ca2+-deficient and normal-condition controls, respectively. The plants were maintained at 25 °C under a 16-h photoperiod with approximately 70 μmol m−2 s−1 light for 10 days. Each treatment (five Ca2+ concentration) consisted of three replicates with 10 plants each in a completely randomized block design. Eight plants were finally selected in each treatment (n = 24). Disease severity was evaluated based on the disease rating scale as described by Zhang and Xu  with mog and Xu .2+, as well as other major macronutrient cations, including Mg2+, Fe2+, Zn2+, Mn2+, Na+, and K+.Spearman’s rank correlation analysis, implemented in SAS9.3, was used to analyze the correlation between tipburn severity and plant endogenous Ca2+ content, SA concentration, and gene expression analysis. Two leaf discs from two rosette leaves were collected, and samples of 18 individuals (36 discs) were pooled together to conduct these experiments at each Ca2+ concentration (n = 18).The in vitro-evaluated plants of inbred lines OQ and QX were used for Ca1BC4 individuals with three different disease rankings: high resistant , tolerant , and high susceptible  from the field test (six plants of each), were used for SA concentration and gene expression analysis. Leaves used in the test were harvested and divided into three types: the outer, middle, and inner leaves, as described in Su et al. .R RT reagent Kit (Takara). Real-time PCR was performed in a Roche thermocycler . Amplification reactions (10 μL) were prepared by mixing 1 μL of cDNA template (250 ng/μL) with 1 × SYBR Green (Roche) and 0.5 μL of each primer at 10 mM. The thermal cycling conditions consisted of an initial denaturation step at 95 °C for 2 min followed by 40 cycles at 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 min, with a final extension step at 72 °C for 5 min. The results were analyzed with Lightcycler version 2.0 (Roche Diagnostics) software. The Chinese cabbage glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal standard to normalize different plant DNA samples , and cDNA was synthesized using a PrimeScripteactions 0 μL wereSamples from the DH population were harvested using the one-eighth-excision method. Individual heads of Chinese cabbage were divided longitudinally into eight equal parts, one of which was then lyophilized and ground to a fine powder for use in the experiments. Analyses were performed with a Dionex ICS3000 ion chromatograph equipped with a high-pressure pump, an ED40 conductometric detector, and a Rheodyne injection valve (25 ml sample loop) following the method of Lee et al. .The aboveground plant parts (∼1 g) were homogenized completely and extracted twice with 3 ml of 90 % methanol. These two supernatants were mixed and dried in a speed vacuum at 40 °C. The residue was then re-suspended in 4 ml of water and incubated at 80 °C for 10 min.−1) was added to 1 mL of the water extract and incubated for 8 h at 37 °C. The sample was then prepared as for free SA.For free SA analysis, 1 mL of the supernatant was re-suspended and incubated for 10 min in 2.5 mL of ethyl-acetate/cyclohexane (1:1) after the addition of 50 μL of concentrated hydrochloric acid (HCl). After centrifugation, the organic phase was dried and then dissolved in 0.5 mL of the high-performance liquid chromatography (HPLC) mobile phase  for HPLC analysis. For total SA quantification (free + bound), 1 mL of β-glucosidase solution   with a flow rate of 0.8 ml minTissue staining with trypan blue was conducted as described by Keogh et al. .−1 Ca2+) after 10 days of cultivation . The affected hydroponic OQ plants were stunted and the leaves were tightly wrinkled, and in some newly developing leaves, the leaf margins and tips became yellow and eventually died . In addition, correlations between the disease severity and the other cations were also not significant at the level of P < 0.05, with the ϱ values of −0.22, −0.12, 0.02, −0.10, 0.09, 0.16, 0.02, and −0.14 for Mn2+, Mg2+, Zn2+, Fe2+, P, K+, and Na+, respectively .Tipburn severity has been reported to be closely associated with the balance between Ca2+ is a major source of calcium for cell growth and signaling. The homeostasis between cytosolic and storage Ca2+ is controlled by a system of membrane-localized Ca2+ pumps and channels, including ECA1-4, ACA4, ACA11, and CAX1. A total of 12 Brassica homologous genes were selected for analysis based on gene annotation of Brassica database  and amino acid sequence alignments. Expression patterns of the 12 genes were assayed and compared between tipburn-susceptible and tipburn-resistant plants of Chinese cabbage . mRNA for both of the CAX1-orthologous genes accumulated to higher levels in the tipburn-resistant line compared to the susceptible line  and resistant lines (QX) after Caine Fig. .1BC4 population that were scored as being either high resistant (HR), tolerant (T), or high sensitive (HS) to tipburn were used for analysis. Leaves used in the test were divided into outer, middle, and inner classes, and the DI of the outer leaves (DI = 88.7) was much higher than that of the inner leaves (DI = 0) , one ECA3 ortholog (Bra031701), one ECA4 ortholog (Bra031593), and an ACA11 ortholog (Bra003276), showed unchanged expression levels  Fig. . In the els Fig. . We alsoels Fig. .2+ transporter genes in the inner leaves was evaluated and compared , and total SA (free SA + SAG) contents. For plants grown in artificial solution, the SA levels in both resistant (QX) and susceptible (OQ) plants showed a generally increasing trend following Caied Fig. . For insent Fig. .Fig. 3Sa−1 FW in healthy HR plants to 6.41, 1.33, and 5.10 μg · g−1 FW in diseased HS plants, respectively , two PAL1 orthologs , and three PAL2 orthologs   and the other requiring phenylalanine ammonia-lyase (PAL)  Fig. .Fig. 4Re2+ deficiency treatment, most genes showed stable expression patterns, although single PAL1 (Bra005221) and PAL2 (Bra003126) orthologs did not  increased, and expression of the other genes was nearly unaffected  showed similar increased expression profiles in both types of leaves, and interestingly, the relative increases in mRNA abundances were higher in the inner leaves compared with the outer leaves Below is the link to the electronic supplementary material.


To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative CaContractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors.2+. Levosimendan, OR-1896 and EMD all increased the sensitivity to β-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α1-AR response.OR-1896 evoked a maximum PIR of 33±10% above basal at 1 μM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89±14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5±5.3%) or milrinone (3.2±4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of β-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca2+ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896.The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca Dowex binds any unconverted cAMP and possible unconverted 5’AMP. Correctional steps take into account the binding ability of Dowex and the conversion efficiency (of [14C]5’-AMP) of Snake Venom.A modified standard two-step phosphodiesterase assay was performed . Frozen 3H]cAMP and [14C]5’-AMP were from PerkinElmer . Cilostamide, rolipram and milrinone were from Tocris . EMD57033 was generously provided by Dr. Norbert Beier of Merck KGaA .Levosimendan was purchased from Zhou Xi Fen Pharm Chemical, Shanghai, China. The identity and purity of this levosimendan was validated by HPLC, MS and proton NMR spectroscopy, as previously described . OR-1896Data are expressed as mean±SEM from n animals unless otherwise specified. P<0.05 was considered statistically significant (Student's t-test and ANOVA). When appropriate, Bonferroni corrections were made to control for multiple comparisons.2+ sensitising agent EMD57033  evoked a PIR on top of OR-1896 alone of 9±3% of basal, and similarly evoked a PIR on top of OR-1896 in the presence of Cil  or Mil . In the presence of Rol and OR-1896, EMD evoked a small PIR of 3±3% of basal; the effect was smaller most likely due to some strips having obtained the maximum PIR (–4 M) given at the end of the experiment demonstrated that all groups had additional capacity to elicit a PIR.We measured the contractile response to OR-1896 in normal rat heart ventricular strips. In the absence of β-AR blockade, OR-1896 evoked a concentration-dependent PIR, reaching a maximum of 33±10% above basal at 1 μM . The PDEimum PIR . IsoprenIn the absence of β-AR blockade in ventricular strips, OR-1896  evoked a PIR similar to that evoked by the PDE3 inhibitors cilostamide  and milrinone   indicative of cAMP-dependency . In rat In the absence of atropine and β-AR blockade in rat ventricular strips, combined OR-1896  and rolipram  evoked a large PIR of 211±33% of basal value . This response was completely reversed by the muscarinic receptor agonist, carbachol  vs. Ctr: 1.8±0.1 mM Ca2+ (n = 7); 2+ sensitizer EMD caused a significant increase in the Ca2+ sensitivity in this preparation (EC50: 1.4±0.1 mM Ca2+ (n = 7), vs. Ctr: 1.8±0.1 mM Ca2+, p<0.05; If OR-1896 was a CaTo verify that OR-1896 was able to inhibit PDEs, we conducted PDE activity assays on homogenized tissue from normal rat heart . OR-1896OR-1896 increased cAMP levels in rat isolated left ventricular cardiomyocytes as detected by the RII_epac cAMP sensor . After a2+ sensitization, it may potentiate effects through receptor systems enhancing Ca2+ sensitivity. Similarly, if OR-1896 operates through a PDE3 inhibitory mechanism, cAMP-dependent receptor signalling should be potentiated or enhanced [1-AR activation) and compared it with levosimendan, which we had studied previously [If OR-1896 exerts effects at least in part through Caenhanced . Therefoeviously .1-AR inhibition, the concentration-response relationship to isoprenaline (β-AR system) was potentiated by OR-1896 (pEC50: OR-1896: 7.6±0.1 (n = 7) vs. Ctr: 7.2±0.2 (n = 7), p<0.05; 2+ sensitizer EMD also sensitized the response to isoprenaline (pEC50: EMD: 7.6±0.1 (n = 7), p<0.05 vs. Ctr; 50: EMD+OR-1896: 7.9±0.1 (n = 7), p<0.05 vs. EMD; In normal rat ventricular strips, in the presence of α50: Lev: 7.8±0.1 (n = 14) vs. Ctr: 7.5±0.1 (n = 14), p<0.05; 50: EMD: 7.9±0.1 (n = 12), p<0.05 vs. Ctr). The combination of EMD and levosimendan further sensitized the concentration-response relationship to isoprenaline beyond that of EMD alone  in the presence and absence of EMD, levosimendan and OR-1896, revealed that only EMD sensitized the response (pEC50 EMD: 5.0±0.1 (n = 14) vs. Ctr: 4.8±0.1 (n = 21), p<0.05) (50 to phenylephrine (pEC50 Lev: 4.8±0.1 (n = 6) vs. OR-1896: 4.9±0.1 (n = 15), both ns vs. Ctr; Stimulation of rat left ventricular strips with phenylephrine (α p<0.05) . Neither2+ sensitivity in the dynamic contractile model used in these studies. In addition, several characteristics of the OR-1896 PIR indicate it is cAMP-mediated. In particular, the PIR is accompanied by a lusitropic response and both responses are sensitive to a reduction of cAMP levels mediated by either muscarinic receptor stimulation (carbachol) or β-AR blockade (timolol). The findings in this study correspond well in all aspects to our prior study on levosimendan in human and rat myocardium [2+ sensitivity [In this paper, we provide strong support that in rat left ventricle the PIR to OR-1896, the active metabolite of levosimendan, results primarily, if not exclusively, from inhibition of PDE3. This conclusion is drawn from the primary findings that 1) OR-1896 did not evoke a PIR in the presence of PDE3 inhibition and 2) OR-1896 did not increase functional Caocardium , furthersitivity ,14.2+ sensitization [2+ sensitization in our model. It is important to clarify with certainty if levosimendan and OR-1896 evoke PIRs through cAMP elevation, because evoking PIRs in this manner is considered detrimental and linked to increased morbidity and mortality in heart failure [The data in this study are consistent with other reports that both levosimendan –5,12,29 tization ,30, we f failure .et al. [et al. show a good correlation between the concentration range in which levosimendan and OR-1896 inhibit PDE3 and the relative force increase in permeabilized guinea pig myocytes. It would have been interesting to know if prior cilostamide treatment would have attenuated the levosimendan and OR-1896 inotropic responses in their models as it did in ours.In ventricular strips where either of two separate PDE3 inhibitors (cilostamide or milrinone) was present, OR-1896 did not evoke a PIR. On the other hand, PDE4 inhibition (rolipram) enhanced the effect of OR-1896. This enhanced PIR is consistent with a previously reported synergistic effect of PDE3 and PDE4 inhibition , and couet al.  that rep2+ sensitivity simultaneously with a PIR in the presence of prior PDE3 inhibition. It is important that such a study is conducted in a model with dynamic handling of Ca2+, such as the one in the present paper. Studies showing Ca2+ sensitization in a steady state model without dynamic Ca2+ handling (e.g. skinned fibres) will not necessarily represent a true physiological effect of levosimendan or OR-1896, thus weakening the translational value to contractility. In this study we found no apparent Ca2+ sensitization of the myofilaments by OR-1896 when concentration-response relationships to Ca2+ were conducted. These findings do not detract from the fact that levosimendan and OR-1896 could sensitize the myofilaments to Ca2+ under certain conditions (e.g. skinned fibres and molecular studies). Furthermore, because we do not measure intracellular levels of Ca2+, we cannot exclude a potential Ca2+ sensitizing mechanism occurring within the cardiomyocyte in our model. However, our results give strong indication that in our model in rat ventricular myocardium, Ca2+ sensitization (if present) does not appear to have any translational value to the PIR. If Ca2+ sensitization of the myofilaments by OR-1896 is sufficient to mediate an inotropic response alone, this inotropic effect should still be present in the presence of PDE3 inhibition. To confirm that our model could measure effects presumed to result from increased Ca2+ sensitivity, we studied EMD 57033. Although OR-1896 and EMD 57033 both bind to troponin C, the latter has been proposed to also bind to a site on myosin that facilitates the actin-myosin interaction possibly altering the ATPase activity [2+ sensitivity of the myofilaments by EMD and OR-1896 is comparable but not identical. Under prior PDE3 inhibition, EMD further increased the PIR and it also sensitized the response to both α1-AR stimulation and the response to cumulative concentrations of Ca2+, neither of which were influenced by OR-1896. This demonstrates at least in part that a potential Ca2+ sensitizer can evoke PIRs which are independent of cAMP accumulation. Thus, even though OR-1896 may increase Ca2+ sensitivity in our model, it does not appear to contribute to the PIR.Studies have established that levosimendan ,10,27 anactivity . This meIt has been reported that combined PDE3 and 4 inhibition increased contractile force in rat ventricle through increasing levels of cAMP . We obseIn our model OR-1896 evoked very similar lusitropic effects as cilostamide and milrinone. This is an important finding and corresponds with other studies in different animal models ,17. The et al. [et al. [2+ sensitivity through phosphorylation of troponin I, which may antagonize the increase in Ca2+ sensitivity induced by OR-1896. In this respect any Ca2+ sensitization by OR-1896 must be more labile than by EMD under the current experimental conditions. Alternatively, levosimendan- and OR-1896-mediated inotropic effects result primarily from increased levels of cAMP. In this case the latter appears to have more support in models directly measuring contractile force, since data are consistent across several different animal models [In our model, activation of the β-AR signalling pathway appears to play a crucial role in the response to OR-1896. Correspondingly, we report that OR-1896 sensitized β-AR stimulation, gave lusitropic effects similar to cilostamide and no inotropic or lusitropic effect in the presence of timolol. This corresponds with other studies; Boknik et al.  reported [et al.  observedl models ,16,18.2+ levels, and although our animal model does not reveal any functional effects beyond PDE3 inhibition, we cannot exclude that OR-1896 enhances Ca2+ sensitivity within the cardiomyocyte. Nonetheless, assuming OR-1896 enhances Ca2+ sensitivity, this mechanism appears to play a negligible role in the OR-1896-evoked PIR; 2) there is a variety of species-dependent variation in the PIR evoked by levosimendan and OR-1896; 3) as such, rat ventricular strips may be unique in that the PIR of OR-1896 appears to be primarily dependent upon enhancing the effect of endogenous noradrenaline. This differs from most other species, in which OR-1896 as well as PDE3 inhibitors elicit their PIRs even in the presence of β-blockers; and 4) the mechanism mediating the PIR of levosimendan and OR-1896 in vivo could be influenced by effects which are independent of both PDE3 inhibition and Ca2+ sensitization [Although our findings support PDE3 inhibition as the mechanism of action responsible for the levosimendan- and OR-1896-evoked PIR, there are some limitations to this study that warrant consideration: 1) we did not measure intracellular Catization ,38. It itization . These eDespite these limitations, the data indicate, at least in rat left ventricular myocardium, that the PIR to both levosimendan and OR-1896 is evoked primarily, if not exclusively, through increasing cAMP levels by inhibition of PDE3.


However little is known about Ca2+ ion channels expressed along peripheral axons and their possible functional significance. The goal of the present study was to test whether voltage-gated Ca2+ channels (VGCCs) are present along peripheral nerve axons in situ and mediate rapid activity-dependent Ca2+ elevations under physiological circumstances. To address this question we used mouse sciatic nerve slices, Ca2+ indicator Oregon Green BAPTA-1, and 2-photon Ca2+ imaging in fast line scan mode (500 Hz). We report that transient increases in intra-axonal Ca2+ concentration take place along peripheral nerve axons in situ when axons are stimulated electrically with single pulses. Furthermore, we show for the first time that Ca2+ transients in peripheral nerves are fast, i.e., occur in a millisecond time-domain. Combining Ca2+ imaging and pharmacology with specific blockers of different VGCCs subtypes we demonstrate that Ca2+ transients in peripheral nerves are mediated mainly by N-type and L-type VGCCs. Discovery of fast Ca2+ entry into the axonal shafts through VGCCs in peripheral nerves suggests that Ca2+ may be involved in regulation of action potential propagation and/or properties in this system, or mediate neurotransmitter release along peripheral axons as it occurs in the optic nerve and white matter of the central nervous system (CNS).In the peripheral nervous system (PNS) a vast number of axons are accommodated within fiber bundles that constitute peripheral nerves. A major function of peripheral axons is to propagate action potentials along their length, and hence they are equipped with Na In addition to Na+ and K+ channels voltage-gated Ca2+ channels (VGCCs) are expressed on peripheral axons. However only few groups have so far directly studied these channels and very little is known about their sub-types, developmental regulation, and function. Ca2+-conductance probably mediated by VGCCs was detected in rat preganglionic cervical sympathetic nerves  repetitive electrical stimulation of rat vagus nerve and of biopsied human sural nerves  VGCCs are present along the axons in several structures including retina  a vast number of axons are accommodated within fiber bundles that constitute peripheral nerves. The major function of peripheral axons is to propagate action potentials along their length, therefore axons are equipped with voltage-gated Na2+ elevations along mammalian peripheral nerve axons in situ under physiological conditions. Answering this question is of great importance for the follow-up research on the functional role of VGCCs in peripheral nerves in situ and in vivo, and is also of clinical and pharmaceutical relevance. Using 2-photon Ca2+ imaging in line scan mode we found that action potentials trigger fast Ca2+ transients along peripheral nerve axons in situ; these Ca2+ transients involve activation of N- and-L-type VGCCs.A major goal of the present study was to test whether VGCCs mediate rapid (in a millisecond time domain) activity-dependent CaC57BL/6N mice were originally obtained from Charles River and bred in house. All experiments were performed in accordance with the guidelines of the Animal Care and Use Committee at the University of Tübingen.2+ ACSF containing in mM: 124 NaCl, 1.25 NaH2PO4, 10 MgSO4, 2.7 KCl, 26 NaHCO3, 2 CaCl2, 2 ascorbic acid, 18.6 glucose. Subsequently nerves were embedded into 2.5% low-melting agarose dissolved in normal ACSF, containing in mM: 124 NaCl, 1.25 NaH2PO4, 1.3 MgSO4, 2.7 KCl, 26 NaHCO3, 2 CaCl2, 2 ascorbic acid, 18.6 glucose and cooled down to 37°C. One hundred micrometres-thin longitudinal nerve slices were prepared on a vibratome , using ice-cold high-Mg2+ACSF. The slices were placed into a Haas-type interface chamber and maintained at room temperature up to 8 h perfused with normal ACSF gassed with 95% O2 and 5% CO2.Newborn mouse pups (P0–2) were sacrificed by decapitation without anesthesia and both sciatic nerves were isolated. The nerves were transferred to a Petri dish and maintained for ~15 min in ice-cold high-Mg2+ indicator Oregon-green-BAPTA-1 (OGB-1 AM) or low-affinity indicator Magnesium Green, as described previously . Four hundred microliter normal ACSF was added to this solution. The final Ca2+ indicator concentration was ~100 μM. A volume of 5 μl of the indicator solution was loaded into a glass micropipette (diameter ~3–6 μm) which was lowered into a nerve slice; a small positive pressure was applied for 10–15 min. Subsequently the slice was washed with ACSF for ~15 min.Individual sciatic nerve slices were loaded with a high-affinity Ca Regehr, . Briefly2+ indicator were transferred to a recording chamber mounted on the stage of a 2-photon laser-scanning microscope  and perfused with ACSF containing 2.5 mM Ca2+. Axons were stimulated with a monopolar glass electrode (3–6 μm tip diameter) filled with normal ASCF. Single pulses  were applied every 30 s using isolated pulse stimulator . To acquire high temporal resolution, line scanning was performed perpendicular to the orientation of the axons, with a frequency of 500 Hz. The dye was excited at 790 nm (Spectra-Physics MaiTai HP Laser) and fluorescence signals were detected using a high sensitivity photomultiplier H7422-40 , after filtering with a DCLP dichroic mirror >500 nm. A laser-scanning system  coupled to an upright microscope  equipped with a 20×, NA 1.1 water-immersion objective  was controlled using ImSpector Pro Software , which also allowed online analysis of the data. The scan head and stimulator were synchronized using Igor Pro 6.2 Software  and an external trigger system .Individual nerve slices filled with Ca2+ fluorescence signal was measured in parts of axonal bundles positioned in the focal plane, as the ratio of the difference between the peak fluorescence and the resting fluorescence (ΔF = F−F0) and the resting fluorescence (F0), after background subtraction. Background region was chosen as the less bright area in the field of view . The analysis was performed using custom-written macros for IgorPro . 10–90% rise-time of Ca2+ transients was measured manually using hairline cursors in IgorPro. To determine the decay time constant, a mono-exponential function was fitted to the decaying part of the transient, from the peak until ~500 ms after the peak. The graphs show mean ± standard error (SEM).The amplitude of the Ca2+ transients were recorded, 3D line-scan pictures (z-stack) of the axons loaded with OGB-1 were recorded. The brightness profile of the line-scans was plotted, and the diameter of single axons was measured in the plane where the axons were in focus. ImSpector Pro Software  was employed for these measurements.To estimate the diameter of single axons within small axonal bundles from which Ca2+ ACSF containing in mM: 124 NaCl, 1.25 NaH2PO4, 10 MgSO4, 2.7 KCl, 26 NaHCO3, 2 CaCl2, 2 ascorbic acid, 18.6 glucose. The nerves were fixed for 1 h in 4% paraformaldehyde (PFA) prepared in phosphate-buffered saline (PBS). PBS contained, in mM: 4.3 Na2HPO4, 1.6 NaH2PO4, 150 NaCl. Afterwards the nerves were washed with PBS (3 times × 15 min) and transferred to 30% sucrose solution in PBS, where they were kept overnight at 4°C. The nerves were then embedded into Tissue-Tek  and frozen at −80°C. Ten micrometre thick slices were prepared with a Leica CM3050S Cryotome and Leica 819 Microtome blades, and transferred onto the glass slides. The slices were washed (3 times × 15 min) with tris-buffered saline (TBS), and incubated in blocking solution for 2 h at room temperature. TBS contained, in mM: 100 Sigma 7–9, 154 NaCl. Blocking solution contained: 3% bovine serum albumin and 0.2% Triton-X in TBS. The slices were incubated with primary antibody overnight at 4°C, washed in TBS (3 times × 15 min), and incubated with secondary antibody coupled to a fluorescent dye for 3–4 h at room temperature. For double and triple immune-labeling the antibodies were applied sequentially, i.e., first primary followed by first secondary, followed by second primary, followed by second secondary, etc. All antibodies were applied in the blocking solution. Washing of slices with TBS (3 times × 15 min) was performed after incubation with each antibody. At the end of the immuno-labeling procedure counterstain 4′,6-diamidino-2-phenylindole  was applied for 5 min at room temperature. The slices were washed with water, dried, covered with Vectashield , and sealed with nail-polish. The list of antibodies used in this study is given in Table Newborn mouse pups (P0–2) were sacrificed by decapitation without anesthesia and both sciatic nerves were isolated. The nerves were transferred to a Petri dish and maintained for ~15 min in ice-cold high-MgAll chemicals were obtained from Sigma  or Carl Roth . OGB-1, Magnesium Green, and pluronic acid were obtained from Invitrogen . Tetrodotoxin (TTX), TTA-P2, ω-conotoxin GVIA, and ω-agatoxin IVa were obtained from Alomone Labs . Stock solutions were prepared according to manufacturer’s instructions and stored at −20°C.T-test. All values are shown as the mean ± SEM. Differences were considered significant at p < 0.05 .Statistical analysis was performed using SPSS statistics Software . Statistical significance of the drug effect was determined with paired-samples 2+ transients occur along mouse sciatic nerve axons in a millisecond time domain, and to assess whether high- or low-affinity indicator works best to measure these transients. We performed 2-photon Ca2+ imaging in nerve slices filled with a high-affinity Ca2+ indicator OGB-1 AM (Kd = 170 nM) or a low-affinity Ca2+ indicator Magnesium Green (Kd = 6 μM), while stimulating axons electrically . This was important, as we observed that at the injected site both Schwann cells and axons took up the dye, while far from the injection site only axons were stained with the indicator and no glial cells were labeled  and 323 ± 30 ms (n = 7), respectively  perpendicular to the orientation of the axons, and tracked changes in the fluorescence of OGB-1 Figure  right ory Figure  right. Tn Figure . Furthery Figure . Ca2+ trs Figure . Based o Regehr, .+ channels. As peripheral nerves contain both TTX-sensitive and TTX-resistant Na+ channels  via the bath. TTX reduced the peak amplitude of Ca2+ transients by 97 ± 6% , suggesting that TTX-resistant Na+ channels and/or Na+-action-potential independent mechanisms may partially mediate evoked Ca2+ increase along sciatic nerve axons.In brain slices, electrical stimulation of gray and white matter axons results in activation of VGCCs located in presynaptic boutons or along axonal shafts . The total divalent concentration was maintained constant by adjusting the levels of Mg2+ ions in the bath. Under these conditions, the peak amplitude of Ca2+ transients was reversibly reduced by 11 ± 2% in 1.8 mM Ca2+ , 29 ± 3% in 1.2 mM Ca2+ , and 55 ± 3% in 0.5 mM Ca2+ . These findings suggest that evoked Ca2+ transients along sciatic nerve axons involve Ca2+ influx from the extracellular space.To investigate the origin of Ca Figures ; n = 3,  Figures ; n = 3,  Figures ; n = 3. 2+ concentration in the bath we observed that relationship between the peak amplitude of Ca2+ transients (ΔF/F) and extracellular Ca2+ concentration is not linear, but Ca2+ influx tends to saturate with increasing Ca2+ level in the bath  at the membrane surface or within the channel pore, to which Ca2+ ions have to bind in order to pass through the channel, get saturated at higher extracellular Ca2+ concentration , a broad-spectrum VGCCs blocker. Cd2+ strongly decreased the peak amplitude of Ca2+ transients, i.e., by 86 ± 2% . We also tested whether application of higher concentration of Cd2+, i.e., 100 μM, results in a stronger inhibition of evoked Ca2+ transients. This was indeed the case, as Ca2+ transients were inhibited by 94 ± 3% p < 0.01 . However, application of 100 μM Cd2+ also caused a steady increase of both basal and peak fluorescence of OGB-1. Increase of Ca2+ indicator fluorescence in the presence of Cd2+ has also been reported in other preparations, e.g., in cultured hippocampal neurons  reduced the peak amplitude of Ca2+ transients by 40 ± 3% . L-type VGCCs blockers nisoldipine (1 μM) or nifedipine (10 μM) inhibited the peak amplitude of Ca2+ transients by 15 ± 3%  or by 12 ± 7% p = 0.32 , respectively. In contrast, application of P/Q-type VGCCs blocker ω-agatoxin IVa (500 nM) or T-type VGCCs blocker TTA-P2 (1 μM) did not significantly change the peak amplitude of Ca2+ transients: amplitude in the presence of ω-agatoxin IVa was reduced by 4 ± 6%  while in the presence of TTA-P2 it was reduced by 4.2 ± 7% . These results indicate that Ca2+ influx along sciatic nerve axons is partially mediated by N- and L-type VGCCs while P/Q and T-type VGCCs are not involved.According to their electrophysiological and pharmacological properties VGCCs are classified into L-, N-, P/Q-, R-, and T-type Dolphin, . L-, N-, Figures ; n = 4.  Figures ; n = 5 o Figures ; n = 4 w Figures ; n = 5. n = 3 animals, Figures n = 3 animals, Figures n = 3 animals, Figures n = 3 animals, Figures n = 3 animals, Figures n = 3 animals, Figures n = 3 animals, Figures To obtain additional independent evidence for the presence of VGCCs in the developing mouse sciatic nerve, we performed immunohistochemistry. We found that both L- and N-type VGCCs were present in the nerve, but their localization was different. L-type VGCCs appeared on bundles of thin axons which often showed weaker labeling with neurofilament (NF200) than the other axons in the nerve  and decay back to baseline with a slower time constant τ of ~320 ms, as estimated with high-affinity Ca2+ indicator OGB-1. These values are quite similar to those obtained with a Ca2+ indicator of comparable Kd in other preparations, including mouse cerebellar mossy fiber boutons  for our experiments. The shortcoming of this experimental design is that OGB-1 may be too slow to precisely follow rapid changes in intra-axonal Ca2+ concentration, and most likely also adds some buffer capacity to the axoplasm  nerve likewise L- or N-type VGCCs were suggested to be of functional significance , may contribute to activity-dependent Ca2+ entry into the axoplasm of peripheral nerve axons. In line with this idea are our findings that Ca2+ transients in mouse sciatic nerve are reduced only partially by specific blockers of VGCCs. Furthermore, also in the unmyelinated nerve fibers of rat vagus nerve neither L- nor N-type nor P/Q-type VGCCs mediated Ca2+ entry along the axons, although Ca2+ transients in that preparation were largely inhibited by Cd2+ , what determines the specificity and the functional output of each Ca2+-dependent process is the amplitude, the time-course and the spatial domain of a transient change in intracellular Ca2+ concentration  and this time probably underestimates the true speed of Ca2+ influx into the axon upon action potential propagation, these Ca2+ transients are well suited to trigger or/and modulate relatively fast axonal processes. For example, transient increase in axoplasmic Ca2+ concentration may be involved in regulation of action potential conduction or frequency through e.g., activation of Ca2+-dependent K+ and/or Cl− channels, or inactivation of Ca2+ channels  along axonal shafts. Rapid (few milliseconds) increases in Ca2+ concentration mediated by VGCCs take place along axonal shafts in white matter of the CNS H-aspartic acid) and stimulated electrically or magnetically  grants: KU2569/1-1 to MK, and PF574/5-1 to FP. This work was also supported by the Werner Reichardt Centre for Integrative Neuroscience (CIN) at the Eberhard Karls University of Tübingen. The CIN is an Excellence Cluster funded by the DFG within the framework of the Excellence Initiative (EXC 307).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.