Both GJIC blockade and Ca2+ mobilization partially depend on ATP release through Cx43 and pannexin (Panx-1) channels, as demonstrated by blocking activity or expression of channels, and inactivating extracellular ATP. By monitoring dye-spreading into adjacent cells, we show that HSP70 released from human monocytes in response to macrophage colony-stimulating factor, prevents the formation of GJIC between monocytes and HMEC. Therapeutic manipulation of this pathway could be of interest in inflammatory and tumor growth.High levels of circulating heat shock protein 70 (HSP70) are detected in many cancers. In order to explore the effects of extracellular HSP70 on human microvascular endothelial cells (HMEC), we initially used gap-FRAP technique. Extracellular human HSP70 (rhHSP70), but not rhHSP27, blocks the gap-junction intercellular communication (GJIC) between HMEC, disrupts the structural integrity of HMEC junction plaques, and decreases connexin43 (Cx43) expression, which correlates with the phosphorylation of Cx43 serine residues. Further exploration of these effects identified a rapid transactivation of the Epidermal Growth Factor Receptor in a Toll-Like Receptor 4-dependent manner, preceding its internalization. In turn, cytosolic Ca Heat shock protein 70 (HSP70) which was initially described as an intracellular protein -3 is alsHigh levels of circulating HSP70 are reported to correlate with monocyte adhesion to endothelial cells . Among t2+ waves.Whereas the role of Cx hemichannels formed by Cx43 in modulation of monocytes-endothelial adhesion is well identified , 23, theExtracellular HSP70 induces signal transduction through the LPS receptor CD14 in monocytes and macrophages , 26. In −1), an index of gap junction permeability. Fig. −1 in untreated to 0.032 ± 0.014 min−1 in rhHSP70-treated cells; mean ± SD, n=8; Fig. −1 in untreated cells to 0.643 ± 0.277 min−1; n=8). Addition of polymyxin B (PMB100 ng/ml) did not prevent the time-dependent decrease in k value observed with rhHSP70 . Thus, the rhHSP70-induced GJIC inhibition is mainly caused by the rhHSP70 protein itself, rather than by any contaminating endotoxin.The effects of rhHSP70 were analyzed on the functionality of gap junctions established between HMEC in confluent monolayer by using the gap-FRAP technique , 35. BriConnexin 43 (Cx43) which is the most widely and highly expressed gap junction protein , is deteAbTLR4)  Fig. .Zonula occludens 1 (ZO-1) is the major protein that interacts with Cx43, precisely through its C-terminal region, to form functional gap junction plaques , 41. IntAbTLR4 antibody while being unaffected by PMB  2 and 4, as well as to promote their association with the epidermal growth factor receptor (EGFR) and the receptor phosphorylation . Since TPMB Fig. .After boiling rhHSP70 solution for 30 min, which denaturizes the protein but not LPS, EGFR phosphorylation was no longer observed, indicating that the effect of intact rhHSP70 was due to HSP70 itself Fig.  and therBy adding an HA motif at its N-terminus, we produced a tagged rhHSP70 that was used to distinguish the exogenous from the endogenous proteins. The purified rhHSP70-HA molecule was a single 70-kDa protein as much functional as the commercially available recombinant molecule . rhHSP70The AKT activation, which is crucial to disrupt GJIC by causing phosphorylation of Ser368 in Cx43 , was rap2+]i in PM-pretreated, fura-2-loaded HMEC , could be prevented by the selective inhibitor of endoplasmic reticulum Ca2+-ATPase, thapsigargin (4μM), suggesting that rhHSP70 recruits Ca2+ from intracellular stores (lower trace). Furthermore, rhHSP70 induced phosphorylation of AKT, ERK and SAPK/JNK within 5–10 min, which was significantly attenuated by the Ca2+ chelator BAPTA/AM , attenuated the Ca2+ from InsP3-sensitive Ca2+-stores. The extracellular application of U-73122 (5 μM), an inhibitor of PLC, reduced both the number and amplitude of rhHSP70-evoked Ca2+ oscillations , decreased both frequency and amplitude of Ca2+ oscillations without affecting the initial peak of Ca2+ release and the basal [Ca2+]i , leading to inositol 1,4,5-trisphosphate (InsP3) formation and release of Caons Fig.  47]. In. In2+ fr+]i Fig. . Pretrea2+]i oscillations while keeping fairly conserved the amplitude of the initial Ca2+ peak  significantly reduced both the frequency and amplitude of i oscillations in nominally Ca2+-free external conditions. As already described in EGF-treated cells i released by InsP3 until a threshold [Ca2+]i is reached to elicit the rapid upstroke  modified with HA Tag in Nter (rhHSP70-HA) has been produced by Proteogenix . The rhHSP70 cDNA was cloned in pT7-MAT-1 expression vector in 2+]i. Briefly, cells were incubated with 2 μM fura-2-AM (40 min at 37°C), then in HEPES-buffered saline solution (HBSS) as previously described [Changes in 340/380 nm ratio of Fura-2 fluorescence were used to measure [Caescribed . Measure3 cells/cm2, growth arrested in FCS-free medium and exposed to apyrase (20 U/ml) or GAP26 (500 μM) or probenecid (100 μM) and/or monocytes stimulated or not by M-CSF (100 nM). Supernatants were collected after 1h to 12h, put on ice and centrifuged at 12,000 g for 10 min.Concentration of ATP in cell media was detected by luciferin-luciferase assay . HMEC were plated at 500 × 103 fluorescent cells were laid on HMEC monolayers. The transfer of dye was visualized after a given time at 37°C. To confirm that the calcein transfer was not due to its non-specific up-take, supernatant collected from donors were added to HMEC. No dye uptake by HMEC was found within 24h.Monocytes were labeled with 4 μM calcein/AM 30 min) together with 10 μM DiL-C18 as previously detailed  min toge. After wHSP70 levels in cell supernatants were evaluated using enzyme-linked immunoabsorbent assay  according to the protocol provided.5 cells per well). After 3-hours, migrated cells were labeled with phycoerythrin-conjugated anti-CD14+ antibody and counted by flow cytometry.HMEC were cultured on 3-μm membrane pores of Transwell inserts until confluency, then FCS-free overnight . 300 μl of monocytes suspension were added  was used to compare data groups of at least five independent experiments. Stimulated samples were compared to controls by two-tailed, unpaired t-tests. *


Vacuolization is an important indicator of pancreatic acinar cell damage. Furthermore, activation of trypsinogen occurs in the endocytic vacuoles; therefore the vacuoles can be considered as ‘initiating’ organelles in the development of the cell injury. In the present study, we investigated the relationship between the formation of endocytic vacuoles and Ca2+ influx developed in response to the inducers of acute pancreatitis [bile acid taurolithocholic acid 3-sulfate (TLC-S) and supramaximal concentration of cholecystokinin-8 (CCK)]. We found that the inhibitor of STIM /Orai channels, GSK-7975A, effectively suppressed both the Ca2+ influx (stimulated by inducers of pancreatitis) and the formation of endocytic vacuoles. Cell death induced by TLC-S or CCK was also inhibited by GSK-7975A. We documented the formation of endocytic vacuoles in response to store-operated Ca2+ entry (SOCE) induced by thapsigargin  and observed strong inhibition of TG-induced vacuole formation by GSK-7975A. Finally, we found that structurally-unrelated inhibitors of calpain suppress formation of endocytic vacuoles, suggesting that this Ca2+-dependent protease is a mediator between Ca2+ elevation and endocytic vacuole formation.The inducers of acute pancreatitis trigger a prolonged increase in the cytosolic Ca 2+ influx and Ca2+-dependent proteases  in the formation of these organelles.Endocytic vacuoles are ‘initiating’ organelles in the development of acute pancreatitis. In the present study, we identified the important roles of store-operated Ca Acute pancreatitis is a frequent disease with considerable human and financial costs ,2. Vacuo2+ responses in pancreatic acinar cells [2+ from the intracellular stores) and prolonged Ca2+ plateau [reflecting the balance of Ca2+ influx and Ca2+ removal by the plasma membrane Ca2+ ATPases (PMCA)] (reviewed in  and the plasma membrane-localized channel-forming protein Orai c) response to supramaximal concentration of CCK (1 nM) resulted in a typical peak-plateau type of response . When applied 30 min before CCK stimulation, 10 μM GSK-7975A did not modify the amplitude of the initial CCK-induced [Ca2+]c peak; the amplitude, normalized to the peak of CCK response, was 100±9% in control  and 101±6% in the presence of GSK-7975A . These experiments indicate that the GSK-7975A is unlikely to modify the ER Ca2+ handling (i.e. Ca2+ leak from the ER and Ca2+ uptake into the ER).The cytosolic Caresponse A. 10 μM  T-cells . The sel T-cells . In our  plateau B and 1C.2+ is frequently used as a substitute for Ca2+ to study influx/current of divalent cations c increase observed in Ca2+-free extracellular solution (2+]c plateau that was formed as a result of Ca2+ addition to the extracellular solution reflects SOCE (2+]c plateau in the acinar cells was very effectively (by approximately 85%) inhibited by GSK-7975A  is capable of inducing the formation of endocytic vacuoles.The treatment of the acinar cells with the sarcoplasmic/endoplasmic reticulum Casolution A. The [Ccts SOCE A. The TGSK-7975A B and 3C.2+ rise to vacuole formation. Calpains (Ca2+-dependent proteases) were recently reported as the effectors responsible for the vacuolization in the acinar cells [2+]c plateau , whereas calpain inhibitor II had no effect on the CCK-induced plateau [Ca2+]c . The similarity between the effect of calpain inhibitors on the CCK-induced formation of endocytic vacuoles suggests that calpain plays an important role in the formation of this type of vacuoles. We then tested the role of calpains in the TLC-S-induced formation of endocytic vacuoles and observed that all calpain inhibitors significantly suppressed vacuole formation. Thus PD150606 at 10 μM concentration significantly suppressed TLC-S-induced vacuole formation  and calpain inhibitor III inhibited vacuole formation by 59±8% . The similarity of the action of different calpain inhibitors suggests that these proteases are important for TLC-S-induced vacuole formation.PD145305 is an inactive analogue of the calpain inhibitor PD150606. The application of PD145305 had no resolvable effect on the vacuole formation when applied at 10 μM  or 30 μM . The CCK-induced formation of endocytic vacuoles was also inhibited by the calpain inhibitor II  and calpain inhibitor III . It should be noted, however, that calpain inhibitor III had a small but statistically significant effect on the [Ca plateau E. Calpai2+ toxicity. We have indeed resolved the inhibition of cell death by 10 μM GSK-7975A and by 10 μM PD150606 . The inhibition was apparent for cells stimulated by CCK  and for cells stimulated by TLC-S . These findings are consistent with the notion that the formation of endocytic vacuoles is an important early step in the development of cell damage triggered by the inducers of acute pancreatitis.The observed inhibition of endocytic vacuole formation by GSK-7975A and by the calpain inhibitors suggests that these compounds could have an overall protective effect against Ca2+ plateaus produced by CCK or TLC-S  are effectively suppressed by the Orai1 inhibitor GSK-7975A and that this is accompanied by a significant reduction in the number of endocytic vacuoles. It is worth noting that GSK-7975A did not completely inhibit the Ca2+ plateau induced by CCK and did not abolish amylase secretion. Vacuolization was also effectively suppressed by structurally unrelated calpain inhibitors, suggesting that these Ca2+-dependent proteases are downstream effectors in the process of Ca2+-induced formation of endocytic vacuoles. The calpain inhibitor PD150606, which was the primary focus of our investigation, did not inhibit Ca2+ influx or amylase secretion.Endocytic vacuoles can be up to 10 μm in diameter and appear specifically in pathological conditions as a result of aberrant retrieval of structures formed by compound endocytosis . The co-in vivo models of this important disease (with the ultimate aim of developing a treatment). The findings that both compounds reduced cell death of CCK- and TLC-S-treated cells but did not abolish a physiological response (amylase secretion) strengthen the case for further in vivo testing of these compounds with the aim of development of a treatment. These further investigations are, however, beyond the scope of the present study.The effective suppression of vacuolization by GSK-7975A and PD150606 indicate that these compounds could be useful for reducing cellular and tissue damage in acute pancreatitis and should therefore be further tested in When the present study was in preparation, a paper from Petersen's group from Cardiff was published . In this


The prognosis of relapsed acute myeloid leukemia (AML) in elderly patients is dismal, even if the AML exhibits a good prognostic karyotype, such as inv(16)(p13.1q22). We present a 72-year-old female with AML with inv(16)(p13.1q22) who suffered five episodes of relapse with temporary complete remission. Maintenance chemotherapy with oral cytarabine ocfosfate hydrate eventually produced persistent molecular complete remission of her AML that had not been induced by conventional regimens including intensive chemotherapy and low dose cytarabine therapy. The high level of tolerability to oral cytarabine ocfosfate hydrate may offer elderly patients with this type of AML a good chance for a cure.The online version of this article (doi:10.1186/s13045-014-0100-6) contains supplementary material, which is available to authorized users. Acute myeloid leukemia (AML) with inv16)p13.1q22) is characterized by a favorable prognosis and good response to treatment with cytarabine [6 after one course of induction chemotherapy comprising behenoyl cytarabine and daunorubicin according to a study protocol  Table 11. HoweveOur patient received lower doses of cytarabine and daunorubicin than the doses that are considered as standard doses for remission induction of AML with inv(16)(p13.1q22) nowadays, and the suboptimal doses of induction chemotherapy may be the cause of her early relapse. However, higher doses of cytarabine and daunorubicin may have put the 72-year-old woman’s life in danger due to associated toxicities. The frail woman eventually went into deep remission after maintenance therapy with a cytarabine prodrug SPAC.SPAC has been shown to be as effective and tolerable as LDAC in treatment of AML -15, thouWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


However, our understanding of PLC in mammalian olfactory sensory neurons (OSNs) is generally limited to its controversial role in odor transduction. Here we employed single-cell Ca2+ imaging and molecular approaches to investigate PLC-mediated Ca2+ responses and its isozyme gene transcript expression. We found that the pan-PLC activator m-3M3FBS (25 μM) induces intracellular Ca2+ increases in vast majority of isolated mouse OSNs tested. Both the response amplitude and percent responding cells depend on m-3M3FBS concentrations. In contrast, the inactive analog o-3M3FBS fails to induce Ca2+ responses. The m-3M3FBS-induced Ca2+ increase is blocked by the PLC inhibitor U73122, while its inactive analog U73433 has no effect. Removal of extracellular Ca2+ does not change significantly the m-3M3FBS-induced Ca2+ response amplitude. Additionally, in the absence of external Ca2+, we found that a subset of OSNs respond to an odorant mixture with small Ca2+ increases, which are significantly suppressed by U73122. Furthermore, using reverse transcription polymerase chain reaction and real-time quantitative polymerase chain reaction, we found that multiple PLC isozyme gene transcripts are expressed in olfactory turbinate tissue in various levels. Using RNA in situ hybridization analysis, we further show expression of β4, γ1, γ2 gene transcripts in OSNs. Taken together, our results establish that PLC isozymes are potent enzymes for mobilizing intracellular Ca2+ in mouse OSNs and provide molecular insight for PLC isozymes-mediated complex cell signaling and regulation in the peripheral olfactory epithelium.Phospholipase C (PLC) and internal Ca Activation of PLC and its associated pathways by cell surface receptors including G-protein coupled receptors and receptor tyrosine kinases, enable cells to react to numerous internal and external signaling molecules, modulators, and stimuli . Upon acr events . In the  animals , in rodeoduction  and thatns OSNs; . Howeveroduction . Adding oduction . Furtheroduction . These r2+, despite one of the major functions of the PLC pathways is to mobilize intracellular Ca2+ to participate in and modulate cellular activity. Also, there is only scattered information about the expression of PLC isozymes  and real-time quantitative polymerase chain reaction (qPCR), we identified multiple PLC isozymes gene transcripts in RNA extracted from the olfactory turbinate tissue and quantified their expression levels. Finally, using RNA in situ hybridization (RISH), we show transcript expression of PLC isozyme β4, γ1, and γ2 in OSNs. Altogether, our data provides physiological and molecular insight for future studies investigating the roles of PLC and its second messenger-mediated signaling pathways in mammalian OSNs.To increase our understanding of the PLC functions in the olfactory system, we first isolated OSNs from the MOE and surveyed PLC activity using a pan-PLC activator and single-cell intracellular CaAdult male and female C57BL/6 background mice of 2–6 months old were used. These mice were in-house bred and were offspring of a transgenic mouse line generated originally in Dr. Robert R. Margolskee’s laboratory, in which the promoter of transient receptor potential channel M5 (TRPM5) drives the expression of GFP . We usedN-2-hydroxyethylpiperazine- N = -2-ethanesulfonic acid buffer (HEPES), 1 MgCl2, 3 CaCl2, 10 Na pyruvate, and 10 D-glucose (pH 7.4 with NaOH). For nominal Ca2+ free bath solution, CaCl2 was omitted from the normal Tyrode’s saline. In some experiments as specified in the text, 1, 2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetracetic acid (BAPTA) was added to a final concentration of 210 μM or 5 mM (for odorants stimulation experiments). PLC activator 2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl) benzenesulfonamide (m-3M3FBS) and its inactive analog o-3M3FBS  were dissolved in dimethylsulfoxide (DMSO) to a stock concentration of 25 mM and freshly diluted immediately before each experiment in bath solution to a final concentration of 15 or 25 μM. The PLC inhibitor U73122 and its inactive derivative U73433 (Tocris) were dissolved in DMSO in stock and diluted in bath solution to a final concentration of 5 or 10 μM. The adenylyl cyclase activator forskolin (EMD Millipore) was dissolved in DMSO in stock and diluted in bath solution to a final concentration of 3 μM. The final concentration of <0.1% DMSO did not induce changes in Ca2+ levels in our Ca2+ imaging experiments. Odor chemicals were purchased from Sigma-Aldrich at the highest purity available. The odor mix used included pentanol, phenethyl alcohol, pentyl acetate, citral, geraniol, isobutyraldehyde, octanal, decanal, hexanal, trans-cinnamaldehyde (10 μM each). Odorants were made by dilution with vigorous vortexing in Tyrode’s saline from 10 mM stock solution stored in 20°C.Chemicals for solutions were purchased from Sigma-Aldrich . Normal bath solution  contained (in mM): 140 NaCl, 5 KCl, 10 2 asphyxiation followed by cervical dislocation and exsanguination through an open heart. The head skin was removed and the nose was split from the midline. The nasal turbinates were dissected and placed in Ca2+- Mg2+- free Tyrode’s saline containing ∼2.5–4 U/ml of papain  and 2 mM cysteine for 2.5–3.5 min at room temperature. Gentle pipetting at the end of enzyme incubation was applied to facilitate cell dissociation. The supernatant was transferred to an O-ring chamber on a cover slip pre-coated with concanavalin A (Sigma).The method of OSN isolation was adapted from our previous study . Briefly2+ levels of OSNs were monitored using Ca2+ sensitive dye Fura-2 as described in our previous publication  is considered to be a stimulus-induced response if the peak value of the change during stimulation was greater than 5% of the resting level and within 30 s after stimulation, The resting Ca2+ level was obtained by averaging 10 data points (3 s each) before applying the stimulus in each cell tested or using the value of the first point before an assumed response if the baseline was stable. In figures containing Ca2+ traces, the vertical scale bar stands for a 50% change in the Ca2+ level measured from the resting Ca2+ level of the recorded region of the same cells.Intracellular Calication , 2011. BTable 1.Oligonucleotide primers were designed against each of the PLC isozymes using NCBI Primer BLAST  or the p2 and olfactory turbinate tissue and positive control tissues including brain, spleen, testis were freshly dissected, homogenized, and total RNA was extracted. For first-strand cDNA synthesis, 500 ng of total RNA template was used and one aliquot (1 μl) of the cDNA reaction was used as the starting template for polymerase chain reaction (PCR). For PLC isozymes that were not expressed at these standard amounts, the cDNA synthesis and PCR steps were repeated with higher amounts of starting material at each step . Additionally, PCR cycles for isozymes that were not expressed with the standard amount of 500 ng of total RNA were increased from 30 to 40 cycles. Templates from cDNA reactions without reverse transcriptase served as negative controls. Resulting amplicons were electrophoretically resolved on 2% agarose gels.Animal euthanasia, tissue dissection and the two-step RT-PCR protocol were described in our previous publication , with miTable 1).The qPCR and data analysis were carried out as previously described . Some ofin situ hybridization protocol including solution making, tissue preparation, riboprobe synthesis, hybridization, and image acquisition have been described in detail in Table 1 for making PLCβ4, γ1, and γ2 RISH probes. Additionally, we adapted a pair of PLCβ4 primers from Allen brain atlas data portal: in situ hybridization data . Procedure for acquiring adult olfactory tubinate tissue from manually deboned noses can be further obtained from our recent video and publication  of PLC isozyme transcripts normalized to expression of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) transcript ± SEM.For comparison of Ca2+ levels mediated by direct PLC activation, we freshly dissociated OSNs from the MOE and stimulated them with the PLC activator m-3M3FBS in intracellular Ca2+ imaging. For our experiments, we selected OSNs that had multiple relatively long cilia. The lengths of the cilia varied, some of which were over 30 μm, indicating the OSNs we recorded were mature . In addition, we also monitored OSN’s excitability by challenging randomly selected OSNs with either a high K+ (40 mM) bath solution and/or forskolin (3 μM). The high K+ solution induces an increase in intracellular Ca2+ levels by depolarizing the cell membrane, which consequently activates voltage-gated Ca2+ channels. Forskolin activates adenylyl cyclase, leading to activation of the canonical cAMP-mediated odor transduction pathway. All OSNs tested responded to high K+ and forskolin  and 81.3% (26 out of 32 OSNs tested from 13 mice) OSNs, respectively, responded to 25 and 15 μM m-3M3FBS . There is no statistical difference between the percent responding cells to the two concentrations . Figure 1D presents the averaged changes in the Ca2+ levels induced by 35 s application of 25 and 15 μM m-3M3FBS, respectively, with significantly higher response amplitude observed at 25 μM . These results indicate that PLC activity is present in a vast majority of mouse mature OSNs and that activation of PLC is a potent mechanism to increase intracellular Ca2+.We freshly made working solution of PLC activator m-3M3FBS from its stock solution (25 mM in DMSO) immediately before each experiment. Bath-application of m-3M3FBS resulted in a large increase in intracellular Ca2+ imaging experiment using the inactive analog o-3M3FBS. o-3M3FBS (25 μM) produced only a slow and very small Ca2+ increase in two out of eight OSNs tested even for longer application time (n = 4 mice), in contrast to the Ca2+ increase evoked by the active activator m-3M3FBS applied for 35 s after to the same cells . The averaged response amplitude is plotted in Figure 2B, showing drastic differences in the response amplitude between these two analogs . To further examine the specificity of the PLC activator in OSNs, we pre-incubated the OSNs with the PLC inhibitor U73122 (5 μM) for 5 min and monitored the effect of U73122 on m-3M3FBS (25 μM)-induced Ca2+ increases. The Ca2+ recording traces and the average response amplitude plot are shown in Figures 2C,D, respectively. U73122 suppressed the m-3M3FBS-induced responses amplitude even after longer m-3M3FBS application time  and the percent responding cells . For control, we repeated the experiment with the U73122 inactive analog U73433 (5 μM). All five cells tested with U73433 responded to m-3M3FBS. The difference in the response amplitude between U73122 and U73433 treatment is statistically significant . Thus, under our experimental conditions, m-3M3FBS activates PLC in OSNs specifically.To test the specificity of m-3M3FBS in OSNs, we performed the same Can = 11 and 18, respectively; Supplementary Figure Additionally, we examined the autofluorescence of m-3M3FBS because a previous study has indicated that the autofluorescence of m-3M3FBS may induce artifacts in the fluorescence measurements . We meas3 and DAG produced as the consequence of PLC activation are capable of increasing intracellular Ca2+ via either internal or external sources in sensory receptor cells -containing Ca2+ free saline. m-3M3FBS was only applied once per cell for 35 s, since subsequent applications of m-3M3FBS often induced smaller responses as compared to the first response (data not shown). As shown in Figure 3A, m-3M3FBS at both 25 and 15 μM induced sizable intracellular Ca2+ increases in both soma and knobs in Ca2+-free bath solution. Also, similar to the m-3M3FBS-induced Ca2+ responses in normal Tyrode’s, high percent of OSNs tested responded to both 25 and 15 μM in Ca2+-free bath solution . There is no statistical difference between the percent responding cells to the two concentrations . In the responses produced by 25 μM m-3M3FBS, the averaged response amplitude measured in the soma in the Ca2+-free bath was very close to the value obtained in the normal Tyrode’s . We did not find any statistical difference between the two values . The average peak value measured from the knobs in the Ca2+ free solution was lower than that measured in the normal Tyrode’s solution , but the difference was not statistically significant . When comparing the 25 μM m-3M3FBS-induced Ca2+ responses in Ca2+ free solution , the difference between soma and knob region is statistically significant . In the responses produced by 15 μM m-3M3FBS, the average response amplitudes from both soma and knobs obtained in the Ca2+ free solution were only slightly reduced as compared to the amplitude from those obtained in normal Tyrode’s , and the difference was not statistically significant . These results indicate that PLC activation-induced increases in Ca2+ levels are primarily due to Ca2+ release from internal stores.Both second messenger IPor cells . We remo2+ levels in a high percentage of OSNs. However, the involvement of PLC in rodent odor transduction remains controversial. In mouse OSNs, Ca2+ increase-mediated by the canonical cAMP olfactory signaling pathway indispensably relies on Ca2+ influx through the CNG channels  to increase the percent responding OSNs. Also, because odor responses attenuate over repeated stimulation and over time, we carefully paired the experiments to ensure data comparable. In control experiments, isolated OSNs were repetitively stimulated with the odor mixture , and the Ca2+ responses were recorded . About 30% OSNs responded to odor mix stimulation in normal Tyrode’s containing 3 mM Ca2+ with a sharp increase in the intracellular Ca2+ levels (35 out 117 cells tested in 19 mice). In general, the peak response amplitude-induced by the odor mixture in the knob was higher than in the soma. When the odor mixture stimulation was repeated, the second response generally was smaller than the first response obtained from the same cells, which is consistent with previous reports . The differences between 1st and 2nd response was not significant . In the knob, the run-down was slightly faster with the average magnitude of second responses being about 75% of the value of first responses. The difference is statistically significant . When the odor mix was applied for the second time in the Ca2+-free saline containing 5 mM BAPTA , five out of six OSNs tested responded with a relatively small, but measurable Ca2+ increase in both the knob and the soma . The amplitude values of the second response measured from the soma and the knob in the Ca2+-free solution were 35 and 26%, respectively, of the first response measured from the same region of the same cells in the normal saline . Interestingly, due to the more reduction in the knob response amplitude in the Ca2+ free saline, the average peak response values for both the soma and knob became similar and not statistically significant . These results demonstrate that a small portion of the Ca2+ increase induced by the odorant mixture in the knob and soma is from internal Ca2+ stores.Our results indicate that activation of PLC represents an important route to change Cachannels . We reas reports . In the 2+ release in Ca2+-free bath by using the PLC inhibitor U73122. Pre-incubation of U73122 (5 μM) significantly reduced or eliminated the Ca2+ response to the 15 s odor mixture stimulation in Ca2+-free saline with stimulation interval ∼540 s  as compared to the response obtained without U73122 pre-incubation . Figure 4G shows the second response amplitude relative to the value of the first response, showing significant difference of the second response amplitude between in Ca2+ free and in U73122/Ca2+ free for both soma and knob region . These data provide further evidence that Ca2+ release via PLC activation is partially responsible for the odorant mixture-induced Ca2+ increases measured from the OSN cell bodies and dendritic knobs.We further examined the role of PLC in the odor mixture-induced CaFigure 5A, top left panel). Positive PCR products of these isozymes as well as other isozymes were obtained from control tissues including brain, spleen, and testis . Because the lack of positive results for other PLC isozymes in the MOE preparations might be due to weak expression, we conducted RT-PCR experiments with increased amounts of RNA as starting template from 500 ng to 1.5 μg and an increased aliquot of the resulting cDNA from 1 to 5 μl for each PCR reaction. We also increased the number of PCR cycles from 30 to 40. Under such conditions, we observed additional positive bands for PLCβ1, β2, γ2 δ1, δ3, δ4, η2 . No product was found for PLCη1 and ζ1 although positive bands with correct sizes were found in control tissue (the size for ζ1 band in the MOE was incorrect). We repeated the experiments in RNA extract from three to six mice and confirmed the identity of all the amplicants by sequencing except δ1 due to the very low yield of the PCR product (Genewiz Inc.). These results indicate expression of multiple PLC isozymes in the olfactory turbinate tissue.To determine PLC gene transcript expression in the MOE, we used RT-PCR to probe all known mouse PLC isozymes. Using total RNA extracted from olfactory turbinate tissues in RT-PCR, we found a strong band of the PLCβ4 amplicant product and moderate or weak bands for PLC isozyme β3, γ1, and ε1 (Gapdh) was used for comparison. We found that PLC β3, β4, γ1, and ε1 were expressed at significantly higher levels than PLC β1, β2, δ1, δ3, δ4, and η2 , with β4 expression being the highest. Additionally, qPCR data also indicate relatively high expression of γ2, which is comparable to the expression levels of β3, γ1 and ε1, although there is no significant difference from the isozymes expressed at lower levels . These quantitative results confirm the expression of multiple PLC isozymes in the olfactory turbinate tissues.We next conducted qPCR to determine the relative expression levels of these isozymes in RNA extracted from the olfactory turbinate tissues. The expression level of a housekeeping gene glyceraldehyde phosphate dehydrogenase . The RISH experiment with the sense probe, which was conducted at the same time and under the same conditions, did not yield any apparent labels . To confirm this result, we repeated the RISH experiment using a different primer set, which sequence information was obtained from the Allen Brain Atlas data portal, to synthesize riboprobes. We obtained the same results (data not shown), indicating that the PLCβ4 mRNA transcripts are expressed in multiple cell types in the olfactory turbinate, which might result in a high expression level as indicated by our qPCR data.Because our qPCR data show that PLCβ4 is expressed at the highest level among all the isozymes and reportedly present in neurons of the cerebellum and retina , we firsFigures 6B,C: low magnification image, Figure 6B’,C’: higher magnification image, from the γ1 and γ2 antisense probes, respectively; Figure 6B″,C″: γ1 and γ2 sense probe labeling, respectively). Further close comparison between the two antisense labeling images, we found that the PLCγ1 signal is apparently restrict to the middle region of the MOE, where mature OSNs are located, while the γ2 signal is present in the entire OSN layer , indicating that the isozyme is most likely expressed in both immature and mature neurons. Taken together, our RISH results confirm the expression of PLCγ1 and additionally, we show presence of PLCβ4 and γ2 in OSNs.We next examined the expression of two PLCγ isozymes because positive immunoreactivity for PLCγ1 is reportedly present in OSNs . We foun2+ imaging provide evidence that direct activation of PLC by m-3M3FBS leads to a large increase in intracellular Ca2+ in a vast majority of OSNs tested. This PLC-mediated Ca2+ increase is primarily from internal Ca2+ release since removal of extracellular Ca2+ has minimal effect on both the response amplitude and percent responding cells. We also showed that in the absence of extracellular Ca2+, the odor mixture-induced small Ca2+ response is mediated by PLC via internal Ca2+ release. Furthermore, we showed expression of multiple PLC isozymes and relatively higher levels of PLC β3, β4, γ1, γ2, and ε1 expression in the MOE as compared to other isozymes. Our RISH analysis confirms that OSNs express at least PLC β4, γ1, γ2 gene transcripts. Taken together, our results reveal the abundance of PLC-mediated activities and multiple isozyme transcript expression in mouse OSNs, providing basic knowledge for further investigation of PLC isozyme-mediated complex cellular signaling and regulations.We have investigated PLC-mediated activity and its isozyme expression in mouse OSNs using physiological and molecular approaches. Our results from single-cell Ca2 to produce second messenger DAG and IP3 , which is made up of a family of ten serine/threonine kinases . Such difference was not observed when stimulated with 15 μM m-3M3FBS . Endoplasmic reticulum (ER), which presumably can serve as Ca2+ stores are present in the knob as indicated by the ER specific markers  the odor mix still induced a small Ca2+ increase in most OSNs tested and the PLC inhibitor U73122 eliminated this response, suggesting a role of PLC and internal Ca2+ release in odor-evoked Ca2+ increase. These results are in consistent with a recent report by 2+ responses in rat cultured OSNs. They found that odor-evoked increase in intracellular Ca2+ persists in the Ca2+-free Rigner’s solution, which is blocked by PLC inhibitor U73122 and this internal Ca2+ release is sufficient to stimulate cGMP production in most OSNs tested. Pathways linking odor stimulation to PLC activation is not established. One possibility is via G-protein Gβγ subunits which are dissociated from the Gαolf upon odor stimulation that activates the canonical pathway. We recently found that the Gβ1γ13 dimer is the dominant Gβγ subunits expressing in the mature OSNs  testing the auto-fluorescence of m-3M3FBS in the bath solution. Our results clearly demonstrate that m-3M3FBS specifically activates PLC in OSNs under our experimental conditions.In this study, we used the PLC activator m-3M3FBS to directly activate PLC because isozyme-specific activators and inhibitors are not available commercially. m-3M3FBS activates all PLC isozymes and has been used in studies of neurons, immune cells, sensory epithelial cells, and various cancer cells. Results of these studies have shown that m-3M3FBS induces intracellular Cativation . Howevertivation  and autotivation . Because2+ and expression of multiple PLC isozyme gene transcripts in a vast majority of mouse OSNs. Because the PLCβ4, γ1, and γ2 that we found in the OSNs can be activated via various cell surface receptors and intracellular Ca2+ influence a variety of cellular activities, we expect diverse and complex roles of PLC isozymes in OSNs. Our study therefore is significant in providing basic knowledge of PLC isozymes for future investigation, especially in the area of regulation of olfactory activities.In summary, we have provided strong physiological and molecular evidence for PLC-mediated changes in intracellular Ca2+ imaging experiment. Tatsuya Ogura and Steven A. Szebenyi designed Ca2+ imaging experiments and performed data analysis. Tatsuya Ogura drafted some Ca2+ imaging results. Aaron Sathyanesan and Abdullah K. AlMatrouk performed RT-PCR, qPCR, and RISH experiments. Aaron Sathyanesan, Abdullah K. AlMatrouk, and Tatsuya Ogura performed qPCR data analyses. Justin Chang performed some Ca2+ imaging and data analysis. Weihong Lin conceived, supervised the project and drafted most of the manuscript. All authors read, edited and approved the manuscript.Steven A. Szebenyi performed most of the CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pleural adhesion (PA) complicates repeated pulmonary surgery.Herein, we prospectively investigated the degree of PA in the second thoracotomy to assess the impact of the previous ipsilateral thoracic procedure.Seventy patients, with a median age of 67 years, undergoing a second thoracotomy with complete medical records of the previous thoracotomy from 4 institutions were included in this study. The mean interval from the first operation to the second operation was 20 months (1-105 months). The site and the extent of adhesion as well as duration and amount of bleeding while dissecting the adhesion were recorded.Fifty-four (76%) patients had PA to the chest wall and 10 (14%) had dense and total PA in the thoracic cavity. Patients with PA experienced more bleeding (215 vs. 29.3 g), a longer drainage period (4.7 vs. 2.3 days), and higher post-operative morbidity (28% vs. 6%) than those without PA in the second operation. According to ROC analysis, cut-off values for operation duration, amount of bleeding, and length of thoracotomy incision in the initial operation for predicting PA were 75 minutes, 10 g, and 6.0 cm, respectively. On the basis of the univariate analysis for PA, operation time >75 minutes, bleeding >10 g, thoracotomy incision length >6.0 cm, and segmentectomy or lobectomy in the initial surgery were significantly associated with PA. Multivariate analysis revealed only thoracotomy incision length >6.0 cm was the independent predictor for PA (p = 0.0065). All patients with thoracotomy incision length >6.0 cm showed PA, but 54% of patients with thoracotomy incision length <6.0 cm. On the other hand, thoracotomy incision length was not associated with dense and total PA. Multivariate analysis identified only post-operative drainage period >5 days in the initial surgery to be the independent predictor for dense and total PA in the thoracic cavity (p = 0.016).Long thoracotomy incision length in the initial surgery is a predictor for PA at the second surgery. Dense and total PA might be caused by post-operative inflammation in the thoracic cavity due to prolonged air leakage.


We argue that the inherent vulnerability of MN to dysregulation of intracellular Ca2+ is deeply associated with discriminating pathogenicity and aberrant crosstalk of most of the critical proteins involved in ALS. Overall, Ca2+ deregulation in MN is at the cornerstone of different ALS processes and is likely one of the factors contributing to the selective susceptibility of these cells to this particular neurodegenerative disease.More than 20 distinct gene loci have so far been implicated in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by progressive neurodegeneration of motor neurons (MN) and death. Most of this distinct set of ALS-related proteins undergoes toxic deposition specifically in MN for reasons which remain unclear. Here we overview a recent body of evidence indicative that mutations in ALS-related proteins can disrupt fundamental Ca Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective degeneration of motor neurons (MN) in the spinal cord, brainstem and cerebral cortex  such as parvalbumin and calbindin , are directly or indirectly involved with Ca2+. It is noteworthy that in such instances, ALS-associated mutations in many of these ALS critical proteins happen to potentiate Ca2+ deregulation and/or result in an increased vulnerability to the effects of Ca2+. For example, VAP-B which is involved in ALS8, is a ER-membrane MAM protein directly engaged in Ca2+ exchange between the ER and mitochondria  driving TDP-43 toxicity across ALS pathology  Glutamatergic excitotoxicity is tough to be mediated by an excessive influx of extracellular ions, including Ca2+, resulting in elevated intracellular Ca2+ levels that can activate cytoplasmatic Ca2+-dependent apoptotic proteins  which promote cell death  account for their mutual involvement in ALS. Interestingly, the so-called “calcium hypothesis” establishing a close link between Ca2+ deregulation and neurodegeneration, has also been suggested to play a central role in other neurodegenerative disorders such as Alzheimer’s, Ataxia, Parkinson’s and Huntington Diseases (Bezprozvanny, 2+ channels and proteins involved in neuronal Ca2+ signalling systems are likely potential targets for therapeutic strategies (Zundorf and Reiser, Overall, we here argue that CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It is a vital nutrient for plant physical design, such as cell wall and membrane, and also serves as a counter-cation for biochemical, inorganic, and organic anions, and more particularly, its concentration change in cytosol is a ubiquitous second messenger in plant physiological signaling in responses to developmental and environmental stimuli. Actin cytoskeleton is well known for its importance in cellular architecture maintenance and its significance in cytoplasmic streaming and cell division. In plant cell system, the actin dynamics is a process of polymerization and de-polymerization of globular actin and filamentous actin and that acts as an active regulator for calcium signaling by controlling calcium evoked physiological responses. The elucidation of the interaction between calcium and actin dynamics will be helpful for further investigation of plant cell signaling networks at molecular level. This review mainly focuses on the recent advances in understanding the interaction between the two aforementioned signaling components in two well-established model systems of plant, guard cell, and pollen.Calcium (Ca Plant signaling transduction can be divided into several steps: the perception of developmental and environmental stimuli, the transduction of cellular signal by complex networks, and the physiological and morphological responses. Numerous ions, molecules, genes, proteins, and physiological processes and biochemical reactions are involved in a typical signaling cascade in a cell  . Biochem2+ is an indisputably important element for both cell structure and signaling during plant growth and development, and also the accumulating data demonstrate its indubitable significance in plant nutrition. Furthermore, recent researches support the concept of cytosolic free Ca2+ ([Ca2+]cyt) as an intracellular second messenger that mediates developmental and environmental stimuli into plant physiological and biochemical responses [2+ as one of the most important regulator in plant signaling have been carried out from whole-plant to single protein levels [2+]cyt is maintained lower than 100 nM in resting plant cells while Ca2+ in the extracellular and organellar levels is at millimolar level [2+ concentration in the cytosol acts as the [Ca2+]cyt signal, which is the result of stimulus triggered Ca2+ influx from extracellular and release from endo-organelles  through Ca2+-permeable channels on plasmamembrane and endomembrane, respectively cyt was shown to trigger the fragmentation of actin bundles in pollen tubes and actin reorganization in guard cells [2+-permeable channel activity and [Ca2+]cyt signal in pollens and guard cells [2+]cyt and actin.Both, guard cells and pollens, are well established model systems for investigation of plant signaling networks, so the similarities and differences between them will give us common signaling characteristics of plant cells, and the accordance of specific cell functioning and signaling. About 20 years ago, the elevation of cyt, the structure or/and dynamics of actin, and to determine the roles of Ca2+ and actin in diverse physiological and developmental processes in guard cell and pollen signaling.Without the aid of developed microscopy, biochemical, and genetic techniques for plant cell investigation, it would be impossible to detect the changes in the levels of [Ca2+]cyt in living plant cells. [Ca2+]cyt concentration change shows complex patterns, such as spiking or oscillation in response to upstream regulators and, thus, to trigger the downstream cellular responses [2+]cyt concentration can be varied from 100 nM (for maintenance of resting cells) to above μM level (during activated state of the cells). As high concentrations of [Ca2+]cyt is toxic to living cells, the elevation of [Ca2+]cyt is brief and/or subcellularly restricted within the physiological range. A set of small-molecules, Ca2+ sensing fluorescent dyes  have been widely used to detect [Ca2+]cyt changes in plant cells cyt, a fluorescent microscope is needed for Ca2+ sensing fluorescent dye application, and FRET-based sensor methods require a confocal microscope.Calcium imaging is the very powerful and visible technique to study the change of cyt rise that, in turn, cause changes of [Ca2+]cyt. Based on above facts, these channels could be regarded as direct upstream elements for generation of [Ca2+]cyt in plant cells. Patch clamp technique enables the recording of Ca2+ channels in plant cells cyt elevation, indicating the opening of plasma membrane Ca2+ channels is essential to initiate the [Ca2+]cyt signaling cyt signals triggered by internal or external stimuli, downstream Ca2+ sensors play vital roles in the perception and decoding of Ca2+ signals. There are four gene families with EF-hand Ca2+ binding domain that have been suggested as Ca2+ sensors in plants, and these families include calmodulin family, calmodulin-like protein (CML) family, Ca2+-dependent protein kinase (CDPK or CPK) family, and the calcineurin B-like protein (CBL) family cyt and alteration of actin structure. For instance, during stomatal movements, Ca2+ signal activation is needed for both opening and closure, and rapid actin rearrangement is also essential for normal stomatal function cyt as a second messenger. In the well-established stomatal movement system, for ABA induced stomatal closing and ABA inhibited stomatal opening processes, [Ca2+]cyt elevation or oscillation could usually be seen, even in Ca2+-independent signaling pathway  [2+]cyt plays a positive regulatory role in ABA activation of pHcyt [2+]cyt elevation has been shown to inactivate inward K+ channels and activate slow anion channels [2+]cyt elevations have been observed cchannels  and, thuchannels . In auxiobserved ,95,96.2, cold shock, ROS, and biotic-stress by pathogens also cause [Ca2+]cyt elevations and stomatal closure [2+ using EGTA abolishes [Ca2+]cyt elevations in response to H2O2, cyclic GMP and hypo-osmolality, indicating plasma membrane Ca2+ influx is essential to initiate the [Ca2+]cyt-mediated signaling [2+ influx through the Ca2+-permeable channels on the plasma membrane for generation of [Ca2+]cyt, and its role in stomatal movements, are response to both abiotic and biotic stresses.In addition to ABA, other abiotic stimuli such as high CO closure ,97,98,99ignaling ,57,100. 2+, but also permeate K+ and are regulated by cyclic nucleotides, calmodulin, and pathogens. The activation of these CNGC channels in response to pathogen generates Ca2+ (Ba2+), current in Arabidopsis guard cell protoplasts, suggesting a possible role of CNG channels in Ca2+-based signaling in this cell type cyt signal [2+ entry mediated by plasma membrane localized Ca2+-permeable channels in guard cells during ABA induced stomatal closing [2+]cyt and actin dynamics are regulated by a complex inter-regulatory loop in guard cells during stomatal movements.Accumulating evidences show that actin is also an active regulator of stomatal movements. Cytochalasin D, which could depolymerize the F-actin, activates K opening . Cytochacurrents , suggestt signal ,50. Acti closing . Altoget2+ also plays important roles in pollen germination and in pollen tube growth, and the crucial effect of this ion during these processes has been described in the 1960s cyt is important for pollen germination, as well as for pollen tube growth and orientation cyt dynamic between non-growing and growing pollen tubes, it has been shown that Ca2+ channels  in the plasma membrane of the pollen tubes are important for regulation of [Ca2+]cyt and tube growth [2+ channels have been identified in both lily pollens and pollen tubes [2+-permeable channels of pollens also have been identified in different plant species [2+]cyt generation cyt generation and tube growth, together with the data from Ca2+-specific vibrating probe and physiological assays by using glutamate receptor like genes, indicating that GLRs are functional Ca2+-channels important for pollen tube elongation cyt in pollen and pollen tube signaling, multiple Ca2+ sensors have been identified that transduce the [Ca2+]cyt signals into physiological and biochemical responses. Growing pollen tubes exhibit higher activity of the CDPK kinase in the apical region, whereas nongrowing tubes show uniform kinase activity. Moreover, the modification of the kinase activity changes the orientation of the tubes, and the increased [Ca2+]cyt enhances the kinase activity that, in turn, leads to reorientation of pollen tube [2+]cyt-concentration distribution cyt [2+ sensors in pollen tubes and were found to be involved in regulating pollen-tube function [2+ signaling to increase the growth rate of pollen-tube tip [2+]cyt. However, up to now, as researchers have limiting knowledge on overall pollen expressing Ca2+ sensors, we need to go a long way to clarify and to identify the downstream components and their interacting partners of Ca2+ signaling network in pollens and pollen tubes.Downstream of [Calen tube . Microinribution .These daCa2+]cyt . CDPK24 function . CPK17 atube tip . These w2+ gradient [2+]cyt in regulation of actin bundling, suggesting that actin might be indirectly regulated by Ca2+ cyt in pollens. In the study by Wang et al., 2004, treatment of F-actin de-polymerization reagents, cytochalasin B and D, activate Ca2+-permeable channels in the plasma membrane of Arabidopsis pollen protoplasts, and also triggers [Ca2+]cyt elevation [2+-channels and [Ca2+]cyt could be repressed by pretreatment of phalloidin, indicating that the regulation of Ca2+ influx through Ca2+-permeable channels is dependent on the actin dynamics and the de-polymerization status of actin is a positive regulator for generation of [Ca2+]cyt cyt .2+]cyt and actin network dynamics are important messengers of signaling in guard cells, pollens, and other plant cells. In this review, we briefly described individual roles of each mentioned messengers and compared the relationship of these two messengers in the two growing plant cell types: the reversible growing model cell, guard cell, and the tip polar growing model cell, pollen. Accumulating data show the existence of the inter-regulation of [Ca2+]cyt and actin in these plant cells, and the importance of interaction between them for the normal function of these cells for the plant life. But the fundamental mechanism for the interaction between these two signaling components is still largely unknown, the [Ca2+]cyt regulation of actin dynamics looks easier to understand, which is primarily dependent on the downstream Ca2+ sensors. In contrast, the actin regulation of [Ca2+]cyt via activation/deactivation of Ca2+-permeable channels is not very clear at molecular level. Is it the reason of abundance of G-actin, or the ratio of G-actin/F-actin in cytosol? In human, gelsolin is important for actin regulation of Ca2+ channels [2+ channel activation and [Ca2+]cyt generation still needs further investigation.During the development and physiological response to external stimuli processes, both [Cachannels ,167. In channels ,168,169,


Natural killer cells have been shown to be relevant in the recognition and lysis of acute myeloid leukemia. In childhood acute lymphoblastic leukemia, it was shown that HLA I expression and KIR receptor-ligand mismatch significantly impact ALL cytolysis. We characterized 14 different primary childhood AML blasts by flow cytometry including NKG2D ligands. Further HLA I typing of blasts was performed and HLA I on the AML blasts was quantified. In two healthy volunteer NK cell donors HLA I typing and KIR genotyping were done. Blasts with high NKG2D ligand expression had significantly higher lysis by isolated NK cells. Grouping the blasts by NKG2D ligand expression led to a significant inverse correlation of HLA I expression and cytolysis in NKG2D low blasts. Furthermore, a significant positive correlation of NKG2D ligand expression and blast cytolysis was shown. No impact of KIR ligand-ligand mismatch was found but a significantly increased lysis of homozygous C2 blasts by KIR2DL1 negative NK cells (donor B) was revealed. In conclusion, NKG2D signaling leads to NK cell mediated lysis of childhood AML despite high HLA I expression. Despite intensification of therapy and the use of new chemotherapeutic agents, one-third to one-half of children with acute myeloid leukemia (AML) experience relapse , 2. HemaThe study was authorized by the ethical institutional review board of the University of Tübingen, Germany. The blasts used in the experiments were isolated from patients who were treated at the Department of Pediatric Hematology/Oncology of the University Children's Hospital Tübingen, Germany. Patients and healthy donors gave informed consent.HLA I typing of the selected cryopreserved AML blasts was provided by the institute for transplant immunology and immunohematology see .Donor A, KIR A haplotype was KIR2DL1, KIR2DL3, KIR2DL4, KIR2DS4, KIR3DL1, KIR3DL2, and KIR3DL3. Donor B, KIR B haplotype was KIR2DL2, KIR2DS2, KIR2DL4, KIR2DS4, KIR3DL1, KIR3DS1, KIR3DL2, and KIR3DL3.http://www.ebi.ac.uk/cgi-bin/ipd/kir/donor_b_content.cgi)  europium release assay. Cryopreserved primary childhood AML blasts and the erythroblastoid cell line K562 (M6 leukemia) were used as target cells. No enrichment of primary childhood AML blasts was done: purity > 80% blasts. K562 was used as positive control to exclude functional NK cell inactivity. Target cells (leukemic blasts) were labeled with 3 ublished . Specifip values < 0.05 were considered significant. Unpaired and paired t-tests as well as Pearson's correlation coefficient were used. For the comparison of donor A and donor B, the pairing considered the blasts ; the comparing condition was the NK activity (specific lysis).Analysis was done using GraphPad Prism Version 5.04. The mean HLA I expression was 416339 ± 55329 molecules per AML blast (range: 35724 to 741642/cell) . AML blan = 14, donors A and B, E : T 10 : 1, without IL2, Pearson r = −0.06, p = 0.83; with IL2, r = −0.11, p = 0.70) .There was no correlation of HLA I expression and NK cell mediated cytolysis of childhood AML blasts independent from E : T ratio and without or with IL2 prestimulation  in donor A and (7 to 7) in donor B with difference neither without nor with IL2 prestimulation Figures . FurthernA = 9, pA < 0.0001; nB = 12, pB = 0.0006) . No differences in susceptibility to NK cell mediated lysis depending on FAB classification were observed.Mean lysis of K562 blasts at E : T ratio 10 : 1 in donors A and B without IL2 prestimulation was 67.2 ± 6.0% and 73.2 ± 4.0% and with IL2 prestimulation was 82.1 ± 4.5% and 81.8 ± 4.7%, respectively. There was no significant difference comparing the lysis ability of donors A and B in any E : T ratio or by integrating the E : T ratios 20 : 1, 10 : 1, 5 : 1, and 2.5 : 1 without and with IL2 prestimulation and there was no difference comparing the increase of lysis by IL2 . IL2 significantly increased the lysis of K562 in both donors   but not with IL2 prestimulation (56.1 ± 6.1% versus 57.6 ± 8.5%). The increase of lysis by IL2 was significant in both donors  but was significantly higher in donor A versus donor B . There was no influence of KIR ligand mismatch between NK donor and AML blasts on specific lysis within each donor. The activating KIR2DS2 and KIR3DS1 encoded by donor B were not found to significantly influence NK mediated cytolysis in this setting of these experiments. Yet, donor B showed a higher lysis of C2 homozygous AML blasts than donor A without and with IL2 prestimulation. The comparison at E : T 10 : 1 was not significant  . No difference was found for C1 homozygous AML blasts  and C1/C2 heterozygous blasts  without and with IL2 prestimulation . The impact of the KIR ligand-ligand model published by the Perugia group (Velardi) in 2002 [In the tested childhood AML blasts, donor B showed significantly higher lysis than donor A ( 0.0029) . At an E = 0.06)  but was NKG2D ligands ULBP1-4 were heterogeneously expressed on the tested AML blasts . The tothigh group was higher than cytolysis of the NKG2DLlow group without IL2 but was not significant at E : T 10 : 1, p = 0.11 . With IL2 prestimulation, no difference between the cytolysis of NKG2DLhigh and NKG2DLlow  was found. Furthermore, we grouped the blasts into HLA Ilow and HLA Ihigh. Blasts with HLA I molecules higher than or within the range of the mean minus standard deviation were defined as HLA Ihigh blasts, whereas blasts with HLA I below the mean expression minus standard deviation were defined as HLA Ilow blasts. Analyzing the HLA Ihigh blasts only, NK cytolysis showed significant positive correlation with NKG2D ligand expression   (Grouping the blasts by the mean MRFI of ULBP1-4 expression (MRFI = 6.6) resulted in two groups with 7 blasts each. The lysis of the NKG2DLp = 0.11 . Compari 0.0076) . Obverse = 0.04) .Improvement in the treatment of childhood AML has led to an overall survival of 60% . Failure in vitro the factors which may influence the NK mediated cytolysis of primary childhood AML blasts in a HLA mismatched model. We found that ALL and AML showed heterogeneous expression of HLA I with significantly higher expression (twice as high) in AML than ALL , and direct cellular interaction inducing unfavorable KIR phenotype and reduction of proactivating NK cell receptors [dull immunophenotype, most likely induced by cellular interaction of AML blasts and NK cells, is reversible during absence of AML blasts in complete remission and is associated with poor outcome, demonstrating the plasticity of NK cell receptor landscape and function by the close interaction with AML [In consequence, there must be other factors leading to sufficient AML blast lysis by NK cells , 25. In eceptors . For inseceptors . Moreovewith AML .low and NKG2DLhigh phenotypes. DNAM-1 ligands were homogeneously expressed (Table  S2) and thus were not suitable to subgroup the blasts and to explain the different lysis of the tested AML blasts, even though DNAM-1 triggering is important in the lysis of several tumor targets including AML [low blasts (high blasts (Two well-characterized and strong mechanisms of NK cell activation are NKG2D- and DNAM-1 signaling . In ALL,ding AML , 32. By ding AML , 25, 30.w blasts . Conversh blasts . In conc in vitro cytolytic differences between donor A and donor B after cytokine stimulation with IL2 and the significant increase in cytolytic activity ex vivo, NK cell transfer in lymphodepletion after chemotherapy or in vivo NK cell stimulation by subcutaneous low-dose and long-term IL2 application should be considered as treatment options in AML patients [ ex vivo expansion [ in vivo expansion plus activation by applied cytokines [These results imply that, for immunotherapeutical approaches recruiting NK cells, a characterization of target cells including HLA I typing and HLA I quantification and characterization of surface markers on the blasts might help estimate how sensitive the targets might be for NK cells. Furthermore, characterization of the donor with regard to HLA typing, KIR genotyping, and KIR RL mismatch can possibly improve donor selection of NK cell immunotherapy or HSCT. According to the minimizedpatients , 33, 34.patients , NKG2D rxpansion , 36 or iytokines , and on ytokines –41. In cThe supplementary material provides additional analyses regarding the impact of HLA E on NK cell mediated cytolysis, comparative analyses of donor A and B taking into account blast HLA I genotype and NKG2DL high versus low expression on NK cytolysis.


In wire myography studies, increasing [Ca2 +]o from 1 mM to 6 mM induced concentration-dependent relaxations of methoxamine pre-contracted rabbit mesenteric arteries. [Ca2 +]o-induced relaxations were dependent on a functional endothelium, and were inhibited by the negative allosteric CaSR modulator Calhex-231. [Ca2 +]o-induced relaxations were reduced by inhibitors of endothelial NO synthase, guanylate cyclase, and protein kinase G. CaSR activation also induced NO production in freshly isolated endothelial cells (ECs) in experiments using the fluorescent NO indicator DAF-FM. Pre-treatment with inhibitors of large (BKCa) and intermediate (IKCa) Ca2 +-activated K+ channels (iberiotoxin and charybdotoxin), and Kv7 channels (linopirdine) also reduced [Ca2 +]o-induced vasorelaxations. Increasing [Ca2 +]o also activated IKCa currents in perforated-patch recordings of isolated mesenteric artery ECs. These findings indicate that stimulation of CaSRs induces endothelium-dependent vasorelaxations which are mediated by two separate pathways involving production of NO and activation of IKCa channels. NO stimulates PKG leading to BKCa activation in vascular smooth muscle cells, whereas IKCa activity contributes to endothelium-derived hyperpolarisations.Stimulation of vascular calcium-sensing receptors (CaSRs) is reported to induce both constrictions and relaxations. However, cellular mechanisms involved in these responses remain unclear. The present study investigates the effect of stimulating CaSRs on vascular contractility and focuses on the role of the endothelium, nitric oxide (NO) and K  In experiments using wire myography, fluorescence microscopy and electrophysiological techniques, we show that CaSRs are expressed in ECs and VSMCs, and that stimulation of CaSRs induces endothelium-dependent relaxations of methoxamine-induced pre-contracted vessels through production of NO and activation of IKCa channels.However, many of the studies examining the functional role of vascular CaSRs have used calcimimetic compounds rather than endogenous CaSR ligands to activate the receptor. Importantly, these agents have also been shown to exhibit off-target actions within the vasculature for example by inhibiting voltage-gated Ca22.1− 1) in accordance with Schedule I of the UK Animals Scientific Procedures Act, 1986. Second-order branches of rabbit superior mesenteric artery were dissected and cleaned of adherent tissue in physiological salt solution (PSS) containing (mM): NaCl 126, KCl 6, Glucose 10, HEPES 11, MgCl2 1.2, and CaCl2 1.5, with pH adjusted to 7.2 with 10 M NaOH. Following dissection, vessels were either cut into 2 mm segments for wire myography studies or were enzymatically dispersed to obtain freshly isolated ECs. To isolate single ECs, vessels were washed in PSS containing 50 μM [Ca2 +]o for 5 min at 37 °C and placed in collagenase solution (1 mgmL− 1) for 15 min at 37 °C. The vessels were then triturated in fresh PSS and the cell-containing solution was collected and centrifuged for 1 min at 1000 rpm. The supernatant was removed and the cells re-suspended in fresh PSS containing 0.75 mM [Ca2 +]o, plated onto coverslips, and left at 4 °C for 1 h before use. To obtain isolated VSMCs, a similar technique was used except that initially the endothelium was gently removed from vessels with a cotton bud, and then the vessels were exposed to an enzyme solution containing collagenase (1 mgmL− 1) and protease (0.2 mgmL− 1) in 50 μM [Ca2 +]o PSS for 15 min.Male New Zealand White rabbits (2.5–3 kg) were killed by intravenous injection of sodium pentobarbitone (120 mgkg2.22/5% CO2) Krebs–Henseleit solution of the following composition (mM): NaCl 118, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, CaCl2 2, D-glucose 10. The mean resting diameter of the vessel segments was 422 ± 19 μm. Once mounted, vessel segments were then normalized to 90% of the internal circumference predicted to occur under a transmural pressure of 100 mmHg23. Mean vessel tensions were 5.3 ± 0.06 mN  in endothelium-intact vessels, and 5.2 ± 0.21 mN  in endothelium removed vessels after normalisation. After normalisation, vessels were left for 10 min and were then challenged with 60 mM KCl for 5 min. Endothelium integrity was then assessed by stably pre-contracting vessels with 10 μM methoxamine. Preliminary experiments demonstrated that 10 μM induced ~ 80% of the maximum methoxamine-induced contraction (data not shown). This was followed by the addition of 10 μM Carbachol (CCh) and vessels in which CCh-induced relaxations were > 90% of pre-contracted tone were designated as having a functional endothelium. When required, endothelium was removed by rubbing the intima with a human hair, and CCh-induced relaxations of < 10% of pre-contracted tone indicated successful removal. Vessel segments were incubated for 30 min in fresh Krebs solution containing 1 mM CaCl2 and then pre-contracted with 10 μM methoxamine, 300 nM U46619, or 60 mM KCl as required. This was followed by cumulative additions of CaCl2, increasing [Ca2 +]o from 1 to 6 mM in the presence of inhibitors tested or their respective vehicles. All inhibitors were added to the vessel segments 30 min before the construction of concentration–response curves to [Ca2 +]o. For each experiment, vehicle controls were performed using vessel segments from the same animal. All relaxant responses are expressed as percentage relaxation of tension induced by either 10 μM methoxamine, 300 nM U46619, or 60 mM KCl. Data points on all graphs are mean values and error bars represent S.E.M. For each experiment n = number of animals, with at least 3–4 vessel segments used from each animal. Responses to increasing [Ca2 +]o were analysed by 2-way ANOVA followed by Bonferroni post hoc tests. p < 0.05 was taken as statistically significant. Bonferroni comparisons are shown above the graph data points whereby: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. controls. EC50 and Emax values were also calculated and compared with Student's t-test. Statistical analysis and graphs were made using Graphpad Prism 6 software .Effects of stimulating CaSRs on vascular tone were investigated using wire myography. Vessel segments of 2 mm in length were mounted in a wire myograph  and equilibrated for 30 min at 37 °C in 5 mL of gassed  for 10 min, washed 4 times with phosphate-buffered saline (PBS), and permeabilised with PBS containing 0.1% Triton X-100 for 20 min at room temperature. Cells were then washed 4 times with PBS and incubated with PBS containing 1% bovine serum albumin (BSA) for 1 h at room temperature. The cells were then incubated overnight at 4 °C with mouse anti-CaSR antibody (1:200) and either goat anti-PECAM-1 (1:200) for ECs or goat anti-α-actin (1:100) for VSMCs in PBS containing 1% BSA. In control experiments, cells were incubated without primary antibodies. The cells were then washed 4 times with PBS and incubated with secondary antibodies conjugated to a fluorescent probe for 1 h (Alexa Fluor 546-conjugated donkey anti-mouse antibody (1:500) and Alexa Fluor 488-conjugated donkey anti-goat antibody (1:500). Unbound secondary antibodies were removed by washing with PBS, and nuclei were labelled with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Sigma-Aldrich). Cells were imaged using a Zeiss LSM 510 laser scanning confocal microscope . Excitation was produced by 543 nm or 488 nm lasers and delivered to the specimen via a Zeiss Apochromat × 63 oil-immersion objective. Emitted fluorescence was captured using LSM 510 software . Two-dimensional images cut horizontally through the middle of the cells were captured and raw confocal imaging data processed using Zeiss LSM 510 software. Final images were produced using PowerPoint .2.42 +]o. The cells were then left for another 30 min at 4 °C to allow complete de-esterification of intracellular diacetate. Inhibitors tested or their respective vehicles were also added at this point. Changes in fluorescence following 5 min of CaSR stimulation with 6 mM [Ca2 +]o were captured using a Zeiss Axiovert 200 M Inverted microscope and processed and analysed using AxioVision SE64 Software . 10 μM CCh was used as a positive control. Additional experiments were CaSRried out to investigate the origins of the baseline fluorescence observed in ECs loaded with DAF-FM diacetate. In these studies, fluorescence changes were recorded 30 min after the addition of the inhibitors tested or their respective vehicles. Changes in fluorescence were quantified by selecting a cell as a region of interest (ROI) and comparing fluorescence levels within the ROI before and after the experimental protocols and analysed using unpaired Student's t-test with p < 0.05 considered significant. Figures and analysis were made using Graphpad Prism 6 .ECs were placed in a sterilised 96-well plate and left for 1 h at 4 °C. Cells were loaded with the nitric oxide fluorescent dye DAF-FM diacetate (1 μM), incubated at 4 °C for 20 min and then washed with PSS containing 1 mM [Ca2.5+ conductances. Recordings were made with an Axopatch 200B amplifier  at room temperature (20–23 °C). Currents were filtered at 1 kHz  and sampled at 5 kHz . Currents were evoked by either dialysing cells with a pipette solution containing 3 μM free Ca2 + or by bath applying 6 mM [Ca2 +]o in whole-cell or perforated-patch recordings, respectively. Additional whole-cell recordings were made with a patch pipette solution containing no added Ca2 +. Current/voltage relationships (I/V) were obtained by applying a 200 ms voltage ramp from − 100 mV to + 100 mV every 30 s from a holding potential of − 60 mV. IKCa currents were calculated from the decrease in current produced by charybdotoxin (CbTx) and the presence of SKCa currents was calculated by any further decrease in current induced by co-application of apamin. I/V relationship are from mean data ± S.E.M., with each point from n = 6 patches and at least 3 animals. Data were analyzed using unpaired Student's t-test with p < 0.05 considered significant. Figures and analysis were made using MicroCal Origin 6.0 software . The external bathing solution for both whole-cell and perforated-patch recordings contained as previously used 2 1, CaCl2 1 (adjusted to pH 7.4 with 10 M NaOH). For whole-cell recordings, the pipette solution contained (mM): KCl 134, HEDTA 5, HEPES 10, MgCl2 5.53 (1 mM free Mg2 +) and CaCl2 0.207 (3 μM free Ca2 +) (pH 7.2). The amounts of MgCl2 and CaCl2 added were determined using EqCal software . For experiments using a pipette solution containing no added Ca2 +, CaCl2 was omitted. For perforated-patch recordings the pipette solution contained (mM): K-aspartate 110, KCl 30, NaCl 10, HEPES 10, MgCl2 1, pH 7.2 with 10 M NaOH, and amphotericin (200 μgmL− 1).Whole-cell and perforated-patch configurations of the patch clamp technique were used to record K2.6Mouse anti-CaSR (SC-47741), goat anti-PECAM-1 (SC-1506), goat anti-α-actin (SC-1616), and secondary antibodies were obtained from Santa Cruz Biotechnology . All other materials were purchased from Sigma-Aldrich  or Tocris . Drugs were dissolved in distilled water or dimethyl sulfoxide (DMSO).33.12 +]o on pre-contracted tone evoked by 10 μM methoxamine. 2 +]o from 1 mM to 6 mM induced concentration-dependent relaxations of rabbit mesenteric artery vessel segments compared to time-matched vehicle controls. 2 +]o, and that physiological plasma [Ca2 +]o levels (1–2 mM) relaxed pre-contracted tone between 30 and 50%. . These data indicate that stimulation of CaSRs exerts vasorelaxant and vasoconstrictor actions via endothelium- and vascular smooth muscle-dependent processes, respectively. In a separate experiment, the same phenomenon was demonstrated using a different vasoconstrictor agent to pre-contract the vessels, namely the thromboxane receptor agonist U46619, with increases in [Ca2 +]o increases in [Ca2 +]o evoking concentration-dependent vasorelaxations that were abolished with the removal of the endothelium .We next studied if removal of a functional endothelium had an effect on [Ca2 +]o-induced relaxations of methoxamine pre-contracted tone were mediated by stimulation of CaSRs, we pre-treated vessel segments with Calhex-231, a negative allosteric modulator of CaSRs 2 +]o-induced relaxations compared to time-matched vehicle controls. In control experiments, 3 μM Calhex-231 had no effect on CCh-induced relaxations of pre-contracted tone  indicating that this calcilytic did not affect the general ability of vessel segments to relax. Consistent with our ideas for an involvement of CaSRs, To examine if [CaThese results provide clear evidence that stimulation of CaSRs induces a profound endothelium-dependent relaxation.3.22 +]o-induced relaxations of pre-contracted tone. 2 +]o-induced vasorelaxations. It is well-established that NO induces vasorelaxations through a pathway involving guanylate cyclase (GC), production of cGMP, and stimulation of protein kinase G (PKG) activity, which subsequently regulates contractile mechanisms in vascular smooth muscle 2 +]o-induced relaxations of pre-contracted tone. Previous studies have shown that PKG may act to suppress EDH mechanisms and that inhibition with KT5823 can evoke significant EDH-mediated vasorelaxations A role for NO has been proposed to mediate actions of CaSRs in the vasculature 2 +]o on NO generation in freshly isolated mesenteric artery ECs using the cell-permeable fluorescent NO indicator DAF-FM. 2 +]o from 1 mM to 6 mM enhanced basal fluorescence by over 30% compared to time-matched vehicle controls, and that these responses were reduced by pre-treatment with 3 μM Calhex-231 and 300 μM L-NAME. 2 +]o were reduced by 3 μM Calhex-231 and 300 μM L-NAME. Positive control experiments revealed that CCh-evoked increases in fluorescence in 1 mM [Ca2 +]o which were unaffected by 3 μM Calhex-231 but as has been shown previously To further investigate if stimulation of CaSRs evokes an increase in NO production, we studied the effect of increasing [CaThese results provide strong evidence that stimulation of CaSRs evokes NO production in mesenteric artery ECs leading to PKG-dependent vasorelaxations.3.3+ channels such as Kv7 and KATP channels expressed in VSMCs may be involved in mediating CaSR-induced vasorelaxations ATP channel inhibitors on [Ca2 +]o-induced relaxations of pre-contracted tone.It is well recognised that IKCa and SKCa channels expressed in ECs have pivotal roles in mediating endothelium-derived hyperpolarisations which couple to relaxation of VSMCs 2 +]o-induced vasorelaxations. Since charybdotoxin (CbTX) blocks BKCa and IKCa channels we investigated the role of IKCa channel activity by pre-treating vessel segments with 100 nM IbTX and 100 nM CbTX. 2 +]o-mediated relaxations compared to IbTX alone. In fact, in the presence of IbTx and CbTx increasing [Ca2 +]o between 1 mM and 5 mM induced an increase in pre-contracted tone . 2 +]o from 1 mM to 6 mM failed to induce relaxations in KCl pre-contracted vessels, and instead, increasing [Ca2 +]o potentiated contractility.These findings suggest that BKCa- and IKCa-mediated hyperpolarisations of VSMCs may make essential contributions to CaSR-evoked vasorelaxations. We tested this proposal by studying the effect of [Ca2 +]o-induced relaxations by using L-NAME to inhibit eNOS and CbTX to block BKCa and IKCa channels. As expected, 2 +]o-induced relaxations of pre-contracted tone. In fact, in the presence of L-NAME and CbTX responses to [Ca2 +]o were similar to those induced in the absence of a functional endothelium , and was inhibited by the non-selective voltage-gated K+ channel blocker tetraethylammonium (TEA). 2 + in the patch pipette solution evoked a much larger mean whole-cell current, which displayed inward rectification at positive potentials, and reversed close to EK. Mean whole-cell currents induced by 3 μM free Ca2 + were reduced by 100 nM CbTX and further inhibited by co-applications of 100 nM CbTX with 100 nM apamin. In these latter conditions, the mean whole-cell current resembled the linear current induced in the absence of Ca2 +. These findings show that rabbit mesenteric ECs express functional native IKCa and SKCa channels as previously described in other native ECs 2 +]o from 1 mM to 6 mM evoked a mean macroscopic current using the perforated patch configuration which had similar inward rectification at positive potentials and reversal potential to the mean whole-cell current induced by 3 μM free Ca2 +. 2 +]o-induced current was inhibited by application of 3 μM Calhex-231 but was not affected by 300 μM L-NAME. Moreover, 2 +]o-induced current was reduced by 100 nM CbTX but was unaffected by 100 nM apamin. These results indicate that stimulation of CaSRs evokes native IKCa currents in rabbit mesenteric artery ECs via a NO-independent pathway.42 +]o induces endothelium-dependent relaxations of pre-contracted rabbit mesenteric arteries. We propose that these CaSR-induced vasorelaxations are mediated by two separate pathways initiated in ECs: NO generation leading to stimulation of a PKG-mediated pathway and opening of BKCa channels in VSMCs, and stimulation of IKCa channel activity which is likely to induce endothelium-derived hyperpolarisations.This study shows that stimulation of CaSRs by increasing [Ca4.12 +]o between 1 mM and 6 mM induced a concentration-dependent relaxation of pre-contracted tone in rabbit mesenteric arteries, which was markedly inhibited by the calcilytic Calhex-231 and abolished by removal of a functional endothelium. In contrast, increasing [Ca2 +]o potentiated pre-contracted tone in endothelium-removed vessel segments. As predicted from these findings, CaSRs were expressed at the plasma membrane of freshly isolated mesenteric artery ECs and VSMCs. These results suggest that in the endothelium-intact artery, CaSR-mediated vasorelaxations are the dominant physiological phenomenon.Increasing [Ca2 +]o has also been shown to induce CaSR-mediated biphasic effects on pre-contracted tone, with an initial contraction followed by relaxation These results are also consistent with earlier studies showing that CaSR mRNA and protein are expressed in ECs 4.22 +]o-induced vasorelaxations of pre-contracted tone were reduced by inhibitors of eNOS, GC, PKG, and BKCa channels. These data provide strong evidence that stimulation of CaSRs induces the well-characterised NO–GC–PKG signalling cascade system, which couples to opening of BKCa channels in VSMCs leading to hyperpolarisation and vasorelaxation. A direct stimulatory action of NO on BKCa channels may also contribute to CaSR-mediated vasorelaxations 2 +]o induced NO production in isolated ECs loaded with fluorescent NO indicator DAF-FM, which was inhibited by Calhex-231 and L-NAME. An interesting observation was that basal DAF-FM fluorescence recorded in the presence of 1 mM [Ca2 +]o was also inhibited by Calhex-231 and L-NAME. This provides evidence that physiological plasma [Ca2 +]o levels may mediate resting vascular tone through stimulation of CaSRs and induce basal production of NO.[Ca2 +] and subsequent NO generation, but only when CaSRs were stimulated with spermine and not with other CaSR agonists such as Ca2 +, Gd3 +, or neomycin. In contrast, this study was able to demonstrate Ca2 +-induced CaSR activation and resulting NO production. Potentially, this difference may reflect variability in CaSR-signalling between cultured ECs and freshly isolated native ECs. In a later study using the calcimimetic AMG 073, Smajilovic et al. + channels Our data are consistent with earlier data indicating a potential role for NO in CaSR-mediated responses in ECs and in other cell types 4.32 +]o-induced vasorelaxations compared to 100 nM IbTX alone. IbTX is a selective BKCa blocker whereas CbTX blocks both BKCa and IKCa, and therefore this additive effect is most likely due to CaSR-mediated activation of IKCa channels which are expressed in ECs but not VSMCs. In addition, increasing [Ca2 +]o from 1 mM to 6 mM evoked macroscopic K+ conductances, which had similar properties to previously described IKCa channels in vascular ECs + channel activity. The selective activation of IKCa over SKCa following CaSR activation potentially reflects the functional co-localisation of IKCa and CaSRs in the EC plasma membrane Co-application of 100 nM IbTX together with 100 nM CbTX produced a markedly greater inhibition of [Ca2 +]o potentiated pre-contracted tone, with a similar magnitude to that observed with removal of a functional endothelium. This further indicates that CaSR-induced endothelium-dependent vasorelaxations are mainly produced through NO- and IKCa channel-mediated pathways. In future studies it will be interesting to investigate if the contribution of these two pathways to CaSR-mediated vasorelaxations differs at different [Ca2 +]o, and with different vascular beds and order branches.Co-application of L-NAME and CbTX produced an additional inhibitory effect on CaSR-induced relaxations compared to applications of L-NAME alone. Moreover, L-NAME did not affect CaSR-induced IKCa channel activity in isolated ECs. These findings indicate that CaSR-mediated vasorelaxations are unlikely to involve interactions between NO-mediated pathways and IKCa channels, which are consistent with earlier findings showing that NO is involved in activating SKCa but not IKCa channels in ECs 2 +]o, as the pan Kv7 blocker linopirdine produce a small by significant reduction in these responses. Heteromeric Kv7.4/7.5 channels and other unidentified Kv channels expressed in VSMCs are reported to mediate relaxations induced by NO Our results also indicate a role for Kv7 channels in CaSR-mediated relaxations at higher o to be much higher within the tight space between the two cell layers than it is within the circulating blood 2 + signalling may then help to amplify the contribution of vascular CaSRs to vascular tone regulation The present work clearly indicates that endothelium-dependent vasorelaxations mediated by CaSRs contribute to the regulation of vascular tone. Indeed even at plasma Ca5Stimulation of CaSRs induces endothelium-dependent vasorelaxations which are mediated by two separate pathways involving production of NO and activation of IKCa channels. NO stimulates PKG leading to BKCa activation in vascular smooth muscle cells, whereas IKCa activity contributes to endothelium-derived hyperpolarisations.British Heart Foundation PhD Studentship for H. Z. E. Greenberg (FS/13/10/30021 to A.P.A); and by the Biotechnology and Biological Sciences Research Council (BB/J007226/1 to A.P.A).This work was supported by a None declared.


To determine if other Ca2+-activated K+ channels (KCa) may participate in this process, mouse kidney and the K+-secreting mouse cortical collecting duct (CCD) cell line, mCCDcl1, were assessed for TRPV4 and KCa channel expression and cross-talk. qPCR mRNA analysis and immunocytochemical staining demonstrated TRPV4 and KCa expression in mCCDcl1 cells and kidney connecting tubule (CNT) and CCD. Three subfamilies of KCa channels were revealed: the high Ca2+-binding affinity small-conductance SK channels, SK1and SK3, the intermediate conductance channel, IK1, and the low Ca2+-binding affinity, BK channel (BKα subunit). Apparent expression levels varied in CNT/CCD where analysis of CCD principal cells (PC) and intercalated cells (IC) demonstrated differential staining: SK1:PC<IC, and SK3:PC>IC, IK1:PC>IC, BKα:PC = IC, and TRPV4:PC>IC. Patch clamp analysis and fluorescence Ca2+ imaging of mCCDcl1 cells demonstrated potent TRPV4-mediated Ca2+ entry and strong functional cross-talk between TRPV4 and KCa channels. TRPV4-mediated Ca2+ influx activated each KCa channel, as evidenced by selective inhibition of KCa channels, with each active KCa channel enhancing Ca2+ entry (due to membrane hyperpolarization). Transepithelial electrical resistance (TEER) analysis of confluent mCCDcl1 cells grown on permeable supports further demonstrated this cross-talk where TRPV4 activation induce a decrease in TEER which was partially restored upon selective inhibition of each KCa channel. It is concluded that SK1/SK3 and IK1 are highly expressed along with BKα in CNT and CCD and are closely coupled to TRPV4 activation as observed in mCCDcl1 cells. The data support a model in CNT/CCD segments where strong cross talk between TRPV4-mediated Ca2+ influx and each KCa channel leads to enhance Ca2+ entry which will support activation of the low Ca2+-binding affinity BK channel to promote BK-mediated K+ secretion.The voltage- and Ca This is accomplished by closely regulating K+ excretion to match K+ intake under normal physiological states. Renal control of K+ secretion occurs predominantly in the late distal tubule, notably the connecting tubule (CNT) and cortical collecting duct (CCD), where K+ secretion is tightly controlled i, in mCCDcl1 cells using standard procedures as done extensively before [2+]i was estimated from the fura-2 fluorescence by excitation at 340 nm and 380 nm and calculating the ratio of the emission intensities at 511 nm in the usual manner every 5 s. Results are subjected to intracellular fura-2 calibration and the ratios converted to [Ca2+]i activity as described by Grynkiewicz et al. [2+]i, on each coverslip with 3–4 coverslips used per treatment group. The data are typically presented as total number of cells analyzed for each treatment group.Ratiometric fluorescence imaging of fura-2 was used to monitor intracellular Cay before , 27. Briz et al. , 27. Typ™ Transwell™ Permeable Supports (0.4 μm pore size) in 24 well plates at 1×105 cells/cm2 in complete growth medium. After 7–9 days, cell monolayer formation in transwell cups was assessed by measuring TEER where all monolayers reached TEER values of 1000 Ω cm2 or more, which was taken as an index of achieving monolayer confluency. Whereupon, the cells were incubated in base medium for another 2 days before performing the experiments. All TEER measurements were made at 37°C in base media. Under these conditions the monolayers displayed an average TEER reading of 3081 ± 155 Ω cm2 (n = 15) during basal conditions.Transepithelial electrical resistance (TEER) measurements were made using an EVOM meter (World Precision Instruments) and chopstick-style electrodes as done previously  and by oThe following chemicals were used in this study: GSK101  from a stock solution (1 mM) in DMSO; apamin  from a stock solution (1 mM) in PBS; iberiotoxin  from a stock solution (0.1 mM) in PBS; TRAM 34 (TOCRIS) from a stock solution (10 mM) in DMSO; and Tertiapin-Q  from a stock solution (10 μM) in PBS.t-test or a one-way ANOVA, followed by the a posteriori Dunnett’s test, as appropriate. For all analysis the significance level was set at P<0.05 unless otherwise indicated in the figure legends.Summary data are presented as mean values ± SEM. “n” is either the number of cells analyzed or the number of sections analyzed as described in the figure legends. Where appropriate, differences among groups of studies were analyzed by either a 2+-activated processes in the collecting duct system. Further, since we have shown that TRPV4 is a key mechanosensitive Ca2+ entry TRP channel in the distal tubule i values  and after activation of TRPV4 (GSK101+). Prior to TRPV4 activation the effect of the blockers on [Ca2+]i was negligible, whereas after TRPV4 activation the 4 blockers reduced [Ca2+]i by 477 ± 45 nM. These results provide support for the concept of a strong cross-talk between TRPV4-mediated Ca2+ influx (GSK+) and activation of the K+ channels.In the absence of TRPV4 activation the addition of the Ki values . In contblockers . The res+ channel blockers on [Ca2+]i were less dramatic, but clearly apparent. This is evident from the representative examples shown for addition of IbTX or TQ alone  can be measured and used to evaluate for activation or inhibition of ion channels if the channels are appropriately expressed at one or both cell borders . All measurements were performed in base media at 37°C  and inhibition (increased TEER) at the cell membranes should be able to be observed.To further evaluate the functional location of TRPV4 and the K37°C see  after th2 under control conditions. Whereupon, all TEER values were normalized to the initial resting TEER value of 1 for all experiments. As shown by the representative example in 2+-activated K+ channels. Whereupon, we tested for the effect of each of the K+ channel selective blockers following TRPV4 activation. As noted, the K+ channel blockers were added to the luminal compartment only after the GSK101-induced decrease in TEER reach a stable low value as indicated by the example in + channel blockers induce an increase in TEER, consistent with inhibition of an active Ca2+-activated K+ channel  is expressed in the collecting duct system (CDS) where it is known to play a key role in controlling K+ secretion in the CNT and CCD i. In contrast, the BK channel, with its lower Ca2+-binding affinity and strategic location at the luminal membrane, would function as the primary effector site for K+ secretion being regulated by TRPV4-mediated Ca2+ influx and the enhanced Ca2+ influx associated with early activation of the SK and IK channels. This model would be consistent with enhance rates of CNT/CCD K+ secretion and K+ wasting during states of high distal tubule flow rates where both TRPV4 and BKα are already established as key players in this process i and flow-dependent K+ secretory rates in the distal tubule and, hence, may play a critical role in generating K+ wasting states associated with inappropriate states of K+ imbalance.In summary, the current studies demonstrates that mCCDcl1 cells and cells of the CNT/CCD express both TRPV4 and an array of KCa channels including small conductance , intermediated conductance (IK1), and large conductance (BK) Ca