Moreover, intradermal (i.d.) administration of the selective µ opioid receptor agonist -enkephalin acetate (DAMGO) attenuated GNTI-evoked scratching behavior, while s.c. administration of DAMGO was ineffective. GNTI provoked c-Fos expression on the lateral side of the superficial layer of the dorsal horn of the cervical spinal cord. Application of 2 Hz EA to LI4 and LI11 decreased the number of c-Fos positive nuclei induced by GNTI. It may be concluded that application of 2 Hz EA to LI4 and LI11 attenuates scratching behavior induced by GNTI in mice and that the peripheral µ opioid system is involved, at least in part, in the anti-pruritic effects of EA.The present study was undertaken to investigate the influence of electroacupuncture (EA) on compulsive scratching in mice and c-Fos expression elicited by subcutaneous (s.c.) administration of a known puritogen, 5′-guanidinonaltrindole (GNTI) to the neck. Application of EA to Hegu (LI4) and Quchi (LI11) acupoints at 2 Hz, but not 100 Hz, attenuated GNTI-evoked scratching. In mice pretreated with the  Itch is a sensation that provokes the desire or reflex to scratch , 2. It iAcupuncture has been used in traditional Chinese medicine for over 2,500 years . PracticSeveral clinical studies have demonstrated the efficacy of acupuncture in alleviating itch. In particular, acupuncture has been shown to relieve histamine-induced itch , type I κ opioid receptor antagonist 5′-guanidinonaltrindole (GNTI) -enkephalin acetate (DAMGO) [This study used the  (DAMGO) . Mice we (DAMGO) ) at the  (DAMGO) , 34. μm) were cut at −25°C with a freezing microtome (Leica CM3050 S). Free-floating sections were stored in PBS at 4°C until immunohistochemistry was performed. Fifteen cervical spinal cord sections from each mouse were randomly selected for immunohistochemistry.The immunohistochemistry procedure was similar to that described by Inan et al. . In brie2O2 to quench endogenous peroxidase activity, washed twice for 10 min with PBS, and blocked with 20% normal goat serum (1 : 20) at room temperature for 2 hr. The sections were then incubated in a shaker for 2 days at 4°C with rabbit c-Fos antibody (1 : 1000 dilution) . After thorough rinsing, sections were incubated in biotinylated anti-rabbit immunoglobulin G secondary antibody  for 2 hr at room temperature. Following two 10 min rinses with PBS, the sections were incubated in an avidin-biotin-peroxidase complex at room temperature for 90 min . Following three 10 min washes in Tris-buffered saline, sections were reacted with 0.05% diaminobenzidine/0.001% H2O2 solution for 4-5 min and washed 3 times for 10 min with Tris-buffered saline. Sections were mounted on slides with 0.25% gel alcohol, air-dried, dehydrated with graded ethanol  followed by xylene (twice for 10 min), and coverslipped with Permount. c-Fos positive nuclei were observed under a light microscope and counted at 200x magnification.Tissues were processed for c-Fos immunoreactivity by the avidin-biotin complex procedure . They weDiaminobenzidine was purchased from Calbiochem ; GNTI, DAMGO, naloxone hydrochloride, and nor-binaltorphimine dihydrochloride were purchased from Sigma Chemical Co. . t-test or one-way ANOVA followed by Tukey's post hoc test. All data are expressed as the mean ± standard error of the mean (S.E.M.). P < 0.05 was considered statistically significant.Statistical comparisons were performed with Student's n = 20), 290.6 ± 36.1 (n = 14), and 398.3 ± 53.7 (n = 14), respectively (P < 0.05), whereas 100 Hz EA pretreatment had no such effect (P > 0.05).To test the effects of EA at two different frequencies (2 and 100 Hz) applied to LI11 and LI4 on GNTI-induced scratching behavior, mice were divided into 3 groups: (1) control, (2) 2 Hz EA, and (3) 100 Hz EA . In contectively . Comparen = 8) and 427.2 ± 55.4 (n = 6), respectively  control, (2) 2 Hz EA applied to bilateral ST36 . At the ectively . The betn = 8) and 347.2 ± 28.8 (n = 17), respectively , animals were divided into 2 groups: (1) naloxone-pretreated controls and (2) naloxone-pretreated plus EA. The scratching behavior was observed for 40 min. After 40 min, the averaged numbers of scratches in the naloxone-pretreated controls and naloxone-pretreated plus EA mice were 318.4 ± 51.6 (ectively . EA did n = 7) and 267.5 ± 41.9 (n = 13), respectively , animals were divided into 2 groups: (1) nor-binaltorphimine-pretreated controls and (2) nor-binaltorphimine-pretreated plus EA. The scratching behavior was observed for 40 min. After 40 min, the averaged numbers of scratches in the nor-binaltorphimine-pretreated controls and (2) nor-binaltorphimine plus EA mice were 452.3 ± 85.9  or DAMGO (10 nmol/site) at the back of the neck. After 15 min, animals were injected with GNTI . After 30 min, the number of scratches was 398.3 ± 53.5 (n = 9) for the control group and 233.1 ± 35.6 (n = 15) for the DAMGO-treated group (P < 0.05) as compared to the control group. To further clarify the roles of peripheral versus central ed group . Thus, in = 11) compared with 425.3 ± 62.8 (n = 7) for the s.c. DAMGO-treated group  and 383.1 ± 62 (n = 10) (P < 0.05).To evaluate the effects of 2 Hz EA on a higher dose of GNTI-induced scratching behavior, the mice were divided into 2 groups: (1) GNTI 0.6 mg/kg (controls) and (2) GNTI 0.6 mg/kg with 2 Hz EA. After 40 min, the averaged numbers of scratches for the control and 2 Hz EA groups were 618.2 ± 78.7 ((n = 10) . PretreaThis series of studies was undertaken to test the effects of EA on GNTI- (0.6 mg/kg) induced c-Fos expression in superficial layers of the cervical spinal cord. Mice were divided into 4 groups: saline control; EA alone; GNTI alone; GNTI plus EA . P < 0.01; Figures P < 0.05; n = 8). GNTI significantly increased the number of c-Fos positive nuclei on the lateral side of the superficial layers of the dorsal horn as compared to the saline control group  are prevented by a μ opioid receptor antagonist. The effect of EA on GNTI-induced scratching may be related to μ, but not κ, opioid receptors.To clarify the relationship between the anti-pruritic effect induced by EA (2 Hz) and opioid receptors, we tested the effects of naloxone and nor-binaltorphimine. In mice pretreated with naloxone, a μ opioid receptor may elicit itch. However, the peripheral μ opioid receptor may have a different role in itch sensation [μ opioid receptors in the anti-pruritic effects of EA, we compared the effects of DAMGO by i.d. and s.c. injection. It is thought that intradermal injection of DAMGO can locally activate peripheral mu-opioid receptors, while s.c. injection of DAMGO can activate both peripheral and central μ opioid receptors. We found that DAMGO (10 nmol/site) attenuated GNTI-induced scratch behavior by i.d. administration but not s.c. administration. The results suggest that only peripheral activation of μ-opioid receptors can inhibit GNTI-induced scratching behavior. Similar results were also observed from another study in that i.d. administration of DAMGO reduced endothelin-1 induced-pruritic behavior [μ receptors on peripheral nerve terminals [μ opioid receptor may be involved in the anti-pruritic effects of low-frequency EA against GNTI-induced scratching.As mentioned above, evidence indicates the central ensation . To furtbehavior . It is aerminals . These rc-fos transcript indicates a population of neurons are activated or excited by nociceptive (noxious) inputs [As mentioned earlier, peripheral receptors uniquely sensitive to pruritogens have yet to be firmly identified. The selectivity of the stimulus appears to be related to a differential connectivity at the spinal level, where a specific class of responsive dorsal horn neurons is located. From there, the stimulus travels via the lateral spinothalamic tract to the thalamus and to the cerebral cortex, where it causes the sensation of itch . The exp) inputs , 63. Noj) inputs  suggeste) inputs , 20–22. ) inputs . In this study, immunohistochemistry was employed to address the issue as to whether a reduction of GNTI-induced c-Fos expression is correlated with EA. The results showed that EA alone did not increase c-Fos expression and that GNTI (0.6 mg/kg) induced c-Fos expression on the lateral side of the superficial layer of the dorsal horn in the cervical cord. Administration of 2 Hz EA to the LI4 and LI11 acupoints decreased the number of c-Fos positive nuclei induced by GNTI. These results further support the contention that EA attenuates noxious stimuli imposed by GNTI. This is the first report demonstrating that EA decreases pruritogen-induced c-Fos expression in a mouse model. Several clinical trials have shown that acupuncture or EA relieves histamine-induced itch , type I μ opioid system is involved, at least in part, in the anti-pruritic effects of EA. Collectively, the application of EA to the LI4 (Hegu) and LI11 (Quchi) acupoints holds promise as an effective anti-pruritic treatment modality.EA diminishes the scratching behavior induced by GNTI in a murine model. The peripheral


Extracellular purines, even non-steroidogenic ones, induced Ca2+-mobilization in the cells, independently of the extracellular Ca2+ concentration. Increases in intracellular Ca2+ concentration induced by extracellular purine agonists were transient, except when induced by ATP or 2MeS-ATP. Angiotensin II (AngII: 100 nM) and dibutyryl-cyclic AMP (db-cAMP: 500 µM) induced both GC secretion and Ca2+-mobilization in the presence of extracellular Ca2+ (1.2 mM). GC secretion by AngII was reduced by nifedipine (10–100 µM); whereas the Ca2+ channel blocker did not inhibit GC secretion by 2MeS-ATP. Thapsigargin followed by extracellular Ca2+ exposure induced Ca2+-influx in H295R, and the cells expressed mRNA/protein of the component molecules for store-operated calcium entry (SOCE): transient receptor C (TRPC) channels, calcium release-activated calcium channel protein 1 (Orai-1), and the stromal interaction molecule 1 (STIM1). In P2Y1-knockdown, 2MeS-ATP-induced GC secretion was significantly inhibited. These results suggest that H295R expresses a functional P2Y1 purinergic receptor for intracellular Ca2+-mobilization, and that P2Y1 is linked to SOCE-activation, leading to Ca2+-influx which might be necessary for glucocorticoid secretion.Purinergic receptor expression and involvement in steroidogenesis were examined in NCI-H295R (H295R), a human adrenal cortex cell line which expresses all the key enzymes necessary for steroidogenesis. mRNA/protein for multiple P1 (A The functional receptors for the extracellular purines (and pyrimidines) in vertebrates are classified into three P1 G protein-coupled receptors  linked to mineral corticoid secretion are well characterized in rats As in BAFC, whose steroidogenic enzyme is closer to the human type than that of the rodent, a human-derived adrenocortical cell line, NCI-H295R cell (H295R) 2+-mobilization. Furthermore, P2Y1 is linked to glucocorticoid secretion. We also have shown that Ca2+ influx is required for glucocorticoid secretion in H295R.In the present study, we have shown that H295R express multiple purinergic receptors, and they are functional for intracellular Ca2A and A2B), P2Y receptors , and P2X receptors (P2X5 and P2X7) were detected . As shown in 1 agonist (100 µM and 1000 µM) significantly increased basal glucocorticoid levels to 64.62±3.54  and 110.87±3.51  respectively, while none of the other purines, pyrimidines, or analogs known as purinergic agonists induced glucocorticoid secretion. A high concentration (100 µM) of adenosine (ADO) significantly reduced basal glucocorticoid levels to 23.11±0.89 (p<0.05).We next examined glucocorticoid secretion by purines, pyrimidines, and their derived analogs. The basal glucocorticoid level (in non-treated H295R) was 40.1±4.147 pmol/10We also placed an order with an analysis laboratory  to quantify basal, 1000 µM 2MeS-ATP-, 500 µM db-cAMP-, and 100 µM forskolin-induced cortisol secretion in the media with HPLC-RIA. The present fluorometric assay and HPLC-RIA were correlated well in the quantification of cortisol . The det4 cells): forskolin (100 µM) to 126.4±9.15; db-cAMP (250 µM) to 87.4±2.36, and AngII (100 nM), to 80.8±12.8,  to 87.4±2.36 (p<0.001) and 82.6+4.94 (p<0.001), respectively. In 0.3 mM Ca2+, 250 µM and 500 µM db-cAMP did not significantly increased the basal levels of glucocorticoid secretion . In 0.6 mM Ca2+, 250 µM and 500 µM db-cAMP did not significantly increase the basal levels of glucocorticoid secretion . In 2.4 mM Ca2+, 250 µM and 500 µM db-cAMP significantly increased the basal level of glucocorticoid secretion to 97.0±5.72 (p<0.001) and 94.5+7.03, (p<0.001), respectively. In 4.8 mM Ca2+, 250 µM and 500 µM db-cAMP significantly increased the basal level to 81.5±3.44 (p<0.001) and 93.3±7.73 (p<0.001), respectively. On the other hand, under Ca2+-free conditions, no significant increase in the basal level was observed. The significance in these differences was analyzed for each point with purine agonists vs base (no agonists) in 2+ concentration with db-cAMP shown in p<0.0001). Thus, extracellular Ca2+ is essential for glucocorticoid secretion in H295R.Forskolin (100 µM), an activator of adenylyl cyclase, dibutyryl-cyclic AMP (db-cAMP), a permeable cAMP analog (250–500 µM), and angiotensin II  were used to confirm glucocorticoid secretion in H295R. All these agents induced glucocorticoid secretion from a basal level of 40.1±4.15  was represented as a ratio in individual cells, and the data were shown as the typical Ca2+-traces with standard deviations (SD) to indicate their variability. The Ca2+ peaks increased from the basal ratio with each stimulant.We have previously reported that UTP, a P2Yin H295R . The int1/2 =  τ) were observed in the Ca2+ trace of each nucleotide  was present. These results indicate that Ca2+-mobilization caused by UTP was due to intracellular Ca2+ release, not to Ca2+ influx from an extracellular site.Some differences in the elimination half lives of the peak ratios  decay of all the purine agonists (1/2 =  τ) of ATP and 2MeS-ATP were 1.28±0.22 min and 2.42±0.14 min, respectively . No τ of the other purine agonists exceeded 1.00 min: ATPγS, 0.79±0.26; ADP, 0.66±0.28; UDP, 0.31±0.16; and BzATP, 0.70±0.21, . Adenosine did not induce a Ca2+ peak  induced a transient peak of Ca2+-mobilization followed by a steady state which was significantly (p<0.05) higher than the basal level (2+ (2+ channels and/or store-operated Ca2+ entry (SOCE) works in H295R. On the other hand, db-cAMP produced a slower Ca2+-mobilization in the presence of extracellular Ca2+  alone produced a quick rise in [Ca2+]i (340/F380) and the τ by the agents represented in Mean±SD were: AngII, 0.44±0.054 and 3.56±0.98 min (N = 7); db-cAMP, 0.42±0.098 (N = 7) and 1.64±0.43 min (N = 5), respectively.AngII is known to act on ATal level . The latevel (2+ . These rlar Ca2+ . No Ca2+ [Ca2+]i . The pea2+ on 2MeS-ATP-induced intracellular Ca2+-mobilization in H295R was done (2+ (1.2 mM Ca2+) existence or Ca2+-free (with 2 mM EGTA) conditions. The peak ratios of the fluorescent intensity (F340/F380) and the τ by the agonist in Mean±SD (N = 14) were: in the presence of Ca2+, 0.64±0.123 and 7.42±2.78 min; in the absence of Ca2+, 0.58±0.164 and 2.82±1.09 min, respectively. The difference between the τ from the two conditions was significant (p<0.001).Comparative assay for the effects of extracellular Cawas done . A 1000 2+ depletion of ER. In the present study, TG (2 µM) significantly increased the Ca2+ free basal ratio from 0.14±0.007 to 0.18±0.011 (p<0.001) transiently .We examined whether extracellular purines induce store-operated calcium entry (SOCE) in H295R in the same way as described in Kawamura et al 2+ increase  was caused by the addition of 1.2 mM CaCl2 to the assay media for a potent Ca2+ rise  and calcium release-activated calcium channel protein 1 (Orai-1) are known to be key molecules for SOCE. Recently, transient receptor potential cation channels C (TRPC) were reported to be necessary for the construction of SOC channels Each image in 33P]-Pi released from [γ-33P]-ATP (33P]-ATP (3.3 µM: 3.0 µCi/mL) for 48 hr. During the incubation, 5 µL of the medium was collected every 2 hr. The collected medium was added into 0.5 mL of PBS containing 50 µg of HCl-activated charcoal , then centrifuged in spin-column at 12,000×g for 1 min. Radioactivity in the extraction (400 µL) was counting using a beta counter. The amount of released [33P]-Pi was expressed as a percentage of the total [γ-33P]-ATP in 5 µL of the medium. ATP metabolism was then estimated.Ecto-ATP activity of H295R was determined by measuring the amount of [33P]-ATP –I. H295RThe graphs in 1 mRNA was employed to show its importance as a target of 2MeS-ATP. In shRNA/eGFP-transfected H295R (2+-mobilization while 2MeS-ATP (100 µM) did not  glucocorticoid secretion, and the present data indicate the possible involvement of SOCE activation.The present study has shown, for the first time, that multiple purinergic receptors are expressed and some of them are linked to CaH295R used in the present study are known as a pluripotent model that has all the key enzymes for steroidogenesis in the three layers of the adrenal cortex , and can produce aldosterone, cortisol, and DHEA s-coupled P1 receptor and Gq/11- or Gi/12-coupled P2 receptors , ecto-5′-nucleotidase CD73 nucleotidase, or ecto-alkaline phosphatase In order to check the activity of ecto-enzymes for ATP and/or ADP, we measured the alternation of ATP or ADP levels (due to metabolization) in the incubation media of H295R. Their metabolizations were observed but the half lives of ATP –II–B wer2+ influx from the extracellular environment through P2X channels is insufficient for glucocorticoid secretion in H295R. The intracellular mechanisms between purinergic receptors and glucocorticoid secretion need to be further investigated.Stimulation by 2MeS-ATP induced at least a half efficacy in glucocorticoid secretion after a 12 hr-incubation . However5 in myenteric neurons 5 transfected into HEK293 cells 2+1. It is well known that GPCR activation induces a transient [Ca2+]i rise, which is independent of extracellular Ca2+ and is caused by intracellular inositol 1,4,5-triphosphate (InsP3) production followed by Ca2+ release from Ca2+ stores 2 activation by ATP or UTP induces InsP3 production and transient [Ca2+]i rise independent of extracellular Ca2+ in BAFC 2+]i rise under Ca2+-free conditions 2+-mobilization in the present study was shown in both the absence and the presence (1.2 mM) of Ca2+, suggesting that this agonist worked via P2Y2, not P2X5, in H295R.Previous studies have reported that 2MeS-ATP activates P2X2+-mobilization with a Ca2+ peak, followed by a lasting phase (higher Ca2+ concentration than the base) in the presence of 1.2 mM Ca2+  did not induce glucocorticoid secretion in the absence of Ca2+ in H295R (2+ influx.Angiotensin II (AngII) is known to induce glucocorticoid secretion in BAFC  mM Ca2+ . BecauseCa2+  Ca2+ influx in glomerulosa cells was described by Gallo-Payet et al 2+ is required for the sustained phase of ACTH-induced steroid secretion. Bird et al. 2+ dependence of 17 alpha-hydroxylase in H295R. In the case of adrenocortical cells, elevation of cAMP can open voltage dependent Ca2+ channels (VDCC) resulting in Ca2+-influx 2+ influx in H295R.UTP induced Ca+ influx . The lacAlthough, in the present study, L-type blocker did not inhibit 2MeS-ATP-induced glucocorticoid secretion but the blocker did reduce glucocorticoid secretion induced by AngII , the con2+ pathways in H295R 2+-mobilization.The present study has also shown the expression of several mRNAs and proteins for the components of SOCE. The expression of multiple Ca2+-mobilizations (release and influx), and the Ca2+ influx is critical for glucocorticoid secretion. These findings also suggest that P2Y1 may be linked to glucocorticoid secretion via SOCE in H295R. Complex purinergic system linked to Ca2+ release and influx, including SOCE, may play an important role in steroidogenesis in the human adrenal cortex.In conclusion, the present study has shown for the first time that H295R express multiple P2 purinergic receptors. Activations of some of them lead to receptor-activated CaDulbecco’s modified Eagle’s medium/Ham’s F-12 medium was obtained from GIBCO Industries, Inc. . Fetal bovine serum (FBS) was purchased from HyClone Laboratories . Antibiotics (penicillin and streptomycin) were purchased from the Meiji Pharma Co. Ltd. . The ITS+ supplement  was obtained from Collaborative Research . ATP, adenosine diphosphate (ADP), adenosine, uridine triphosphate (UTP), uridine diphosphate (UDP), and 2-(methylthio) adenosine 5′-triphosphate (2MeS-ATP) were purchased from Sigma-Aldrich, Inc. . Adenosine-5′-O-(3-thiotriphosphate) (ATPγS) was obtained from Enzo Life Sciences, Inc. . All other chemicals were of analytical grade.NCI-H295R cells Total RNA was isolated; digestion of DNA fragments in the isolated RNA, and purification of the RNA was performed using Total RNA Isolation . Following spin column purification, RNA samples were reverse-transcripted to cDNA using the PrimeScript II 1st strand cDNA synthesis kit  with oligo-dT . The cDNA then underwent PCR using QuickTaq HS DyeMix  for 33 cycles. PCR products were subjected to gel electrophoresis on a 2.0–2.5% agarose gel, depending on the expected molecular size of the PCR products. PCR conditions were identical for all primers .H295R were lysed in RIPA Buffer  with protease inhibitor cocktail . The crude lysate was treated to remove the extraction buffer with the Pierce SDS-PAGE Sample Kit . The extracted protein samples (2.8–3.3 mg protein/mL) were mixed with the same volume of the EzApply Sample Buffer  containing dithiothreitol (100 mM) and were heated at 98°C for 5 min (37°C for 15 min in some cases depending on the target protein). The heat-treated samples were applied to the Perfect NT Ge (5–20% gradient), a precast gel  for PAGE (200 V – 35 min). Following PAGE, the protein samples were transferred to the ClearBlot, a PVDF membrane  by Western blotting (400 mA – 60 min).Membranes containing transferred proteins were treated with bovine serum albumin (5%) for 1 hr at room temperature. Following blocking, the PVDF membrane was cut into pieces along the locus lanes of the LandMark Broad-range Prestained Protein Marker , a pre-stained molecular marker. These rectangular membrane strips were treated with each primary antibody diluted in Solution-I of the CanGetSignal System , the immunoreaction enhancer solution, for 1 hr at room temperature. After washing with TBS-Tween (0.05–0.1%), the membrane pieces were reacted with 2ndary antibodies in Solution-II of the CanGetSignal System for 1 hr at room temperature. Washed membranes were then dumped into EzWetLumi plus Solution , a highly sensitive substrate for HRP, to detect the developing chemiluminescence signals for the HRP-conjugated 2ndary antibodies that bind to the primary antibodies on the PVDF membranes. X-ray films  were exposed to the PVDF membranes, and were developed by a film developing system . The developed films were captured by a scanner system  to detect the bands on the PVDF membranes. According to the explanation appendixes of the antibodies used, multiple signal bands were occasionally expected. In the case of multiple bands, the control peptide for the antibody was used for competitive inhibition. Supporting details are in the 2+-free, phosphate buffered saline containing 0.5 mM EGTA (PBS-EGTA), followed by washing once with HamF12/DMEM medium. The washed cells were incubated at 37°C for appropriate hrs in a total incubation volume of 1 ml in a humidified atmosphere of 5% CO2 in air. At the end of the incubation, 0.5 ml of each incubation medium was removed for the corticosteroid assay. Then the cells were detached for a 10 min incubation period at 37°C in 0.1% trypsin containing PBS-EGTA to count the cell number by a hemocytometer. The removed media for assay was mixed with CH2Cl2 to extract corticosteroid. The corticosteroid in CH2Cl2 was transferred to the sulfuric acid reagent (H2SO4 : ethanol = 7∶ 3) and mixed well. The emission of 520 nm excited by 390 nm was measured to quantify corticosteroid using cortisol as a standard The adherent cultured H295R were washed twice with Ca2+ concentration ([Ca2+]i) measurements. The cells were loaded with 5 µM fura-2 acetoxymethylester (fura-2/AM). The fluorescence was monitored at an emission wavelength of 510 nm by the imaging system operated by The AquaCosmos . The [Ca2+]i was shown as the ratio of the 510 nm fluorescence intensity excited at 340 nm, to that at 380 nm.H295R cells that had been cultured for one day on cover slips treated for cell adhesion , were used for intracellular Ca1) for RNAi. A cassette for the target was made by annealing the top and the bottom, followed by cloning the double strand DNA into pENTR/U6 using T4 DNA ligase . Finally we selected two different targets in P2Y1 mRNA and made two different cassettes. The top strand of cassette-1, the bottom strand of cassette-1, the top strand of cassette-2, and the bottom strand of cassette-2 were: CACCGCTGTCTACATCTTGGTATTCCGAAGAATACCAAGATGTAGACAGC, AAAAGCTGTCTACATCTTGGTATTCTTCGGAATACCAAGATGTAGACAGC, CACCGCCACGTATCAGGTGACAAGACGAATCTTGTCACCTGATACGTGGC, and AAAAGCCACGTATCAGGTGACAAGATTCGTCTTGTCACCTGATACGTGGC, respectively. A set of the top and the bottom strands of the DNA was made. The plasmid was transformed using the One Shot TOP10 Chemically Competent E. coli  and incubated at 37°C in LB followed by spreading on an LB agar plate with kanamycin (50 µg/mL) at 37°C overnight. Some colonies were selected and amplified in LB with kanamycin. Amplified plasmids were extracted and purified to use in transfection. Transfection was done in suspended H295R by electroporation with electroporator MP-100  and eGFP plasmid was co-transfected with the shRNA plasmid. As a control, a plasmid for a non-coding cassette was used and eGFP plasmid was again co-transfected. Each plasmid (0.5 µg) was added to 105 cells in 10 µL electrode buffer  followed by pulsation (1800 V – 20 msec).To generate the vector for short hairpin RNA (shRNA ) for use in RNA interference (RNAi), pENTR/U6 plasmid  with a DNA cassette for shRNA was used. The cassette for shRNA was composed of double strand DNA. Each DNA strand was called either ‘top’ or ‘bottom’. We checked the sequences for RNAi using the program on Invitrogen’s site and selected the sequence of the target mRNA (P2Y2+ measurements) and data were analyzed by one- or two-way analysis of variance (ANOVA) followed by Tukey-Kramer post-hoc tests in order to reduce the false positive or type I error rate. A p-value of less than 0.05 was considered to be statistically significant.Values are expressed as Mean±SE or Mean±SD . In these tests, basal levels, those stimulated by 1000 µM 2MeS-ATP, by 500 µM db-cAMP, and by 100 µM forskolin were compared between the fluorometric analysis and HPLC-RIA (N = 4–6).(TIF)Click here for additional data file.Figure S2Analysis of expression of ecto-alkaline phosphatase. Images of ALP (1) and TRPC5 (2) on the H295R cell surface. 1A and 2A: transparent images, 1B and 2B: images of Alexa 488 as protein expression, 1C and 2C: images of DAPI for nucleoli, and 1D and 2D: overlay of A, B, and C. For DyLight 488-labeled 2ndary antibody (Alexa Fluor 488) examination, cells were excited at 488 nm and the emission was observed through a 520 nm band path filter, respectively. For DAPI, the cells were excited at 358 nm and the emission was observed through a 460 nm band path filter. TRPC5 (2B) expression is positive but ALP is not (1B) as ecto-ALP. All images were originally observed as monochromes and changed to pseudo-colors. Scale bar indicate 50 µm.(TIF)Click here for additional data file.Figure S3Time-dependent cortisol secretion assay in H295R. A) Time-course of cortisol secretion in H295R. Time course study of cortisol secretion by 1 mM 2MeS-ATP in H295R. Data represent the Mean±SE (N = 4). The curve fitting in A was performed by the GraphPad Prism . B) Cortisol secretion following short-term incubation in H295R. Short-term incubation procedures were performed using 2MeS-ATP. The media was removed from the well at 1, 2, 4, 8, 12, 24, or 36 hr after application of the agents. Once the media was removed, the wells were washed and refilled with the same volume (1 mL) of fresh media and maintained in incubation without any agents until 48 hr. Graph shows cortisol secretion at 48 hr after the application. Data represent the Mean±SE (N = 4).(TIF)Click here for additional data file.Figure S42+-free conditions in H295R.Effects of purinergic agonists on GC secretion under Ca No agonists tested under Ca2+-free conditions (2 mM EGTA) induced significant GC secretion. For comparison, GC secretion by 2MeS-ATP under standard condition (1.2 mM Ca2+) is shown as Mean±SE (N = 4).(TIF)Click here for additional data file.Figure S5Comparative assay for the effect of an L-type VDCC blocker on 2MeS-ATP- or AngII-induced glucocorticoid secretion. Effects of nifedipine, an L-type VDCC-blocker, on 2MeS-ATP or AngII-induced glucocorticoid secretion in H295R. The cells were incubated at 37°C for 48h. Each histogram represents the Mean±SE (N = 4). ‘*’, ‘**’, statistical significance at p<0.05, p<0.01, respectively.(TIF)Click here for additional data file.Table S1Sensitivity of cortisol assay in the fluorometric analysis and HPLC-RIA.(DOC)Click here for additional data file.Table S2Interassay coefficient of variations (CVs) in the fluorometric analysis for cortisol (N = 4–6).(DOC)Click here for additional data file.Table S3Interassay coefficient of variations (CVs) in HPLC-RIA for cortisol (N = 4).(DOC)Click here for additional data file.Table S4Gene specific primers used for PCR amplification.(DOC)Click here for additional data file.Text S1Supporting Information.(DOC)Click here for additional data file.


Previous research has documented the molecular coupling of small-conductance Ca2+-activated K+ channels (SK channels) to VDCCs in mouse cardiac muscle. Little is known regarding the role of RyRs-sensitive Ca2+ release in the SK channels in cardiac muscle. In this study, using whole-cell patch clamp techniques, we observed that a Ca2+-activated K+ current  recorded from isolated adult C57B/L mouse atrial myocytes was significantly decreased by ryanodine, an inhibitor of ryanodine receptor type 2 (RyR2), or by the co-application of ryanodine and thapsigargin, an inhibitor of the sarcoplasmic reticulum calcium ATPase (SERCA) . The activation of RyR2 by caffeine increased the K,CaI in the cardiac cells . We further analyzed the effect of RyR2 knockdown on K,CaI and Ca2+ in isolated adult mouse cardiomyocytes using a whole-cell patch clamp technique and confocal imaging. RyR2 knockdown in mouse atrial cells transduced with lentivirus-mediated small hairpin interference RNA (shRNA) exhibited a significant decrease in K,CaI (p<0.05) and [Ca2+]i fluorescence intensity (p<0.01). An immunoprecipitated complex of SK2 and RyR2 was identified in native cardiac tissue by co-immunoprecipitation assays. Our findings indicate that RyR2-mediated Ca2+ release is responsible for the activation and modulation of SK channels in cardiac myocytes.In cardiomyocytes, Ca These channels link the intracellular calcium concentration to a wide variety of cellular processes 2+ ions are derived from the influx of Ca2+ into the cell through voltage-dependent Ca2+ channels (VDCCs) and by the release of Ca2+ from internal Ca2+ stores SK channels are voltage-insensitive and Ca2+ functions as a second messenger and mediates its own release from internal stores through the activation of ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3R) 2+ from an intracellular Ca2+ storage compartment, the sarcoplasmic/endoplasmic reticulum (SR/ER) 2+ release from intracellular Ca2+ stores plays a major role in mammalian Ca2+ signaling triads formed by voltage-gated Ca2+ channels, RyRs, and SK channels in neurons 2+ channel in cardiac tissue 2+ release channel in the regulation of the SK2 channel in mouse atrial myocytes using electrophysiology and the lentiviral-mediated delivery of small interference RNA (siRNA) against RyR2 to cardiomyocytes. Our study is the first to exhibit functional modulation of RyR2-mediated Ca2+ release on the SK2 channel in cardiac myocytes and describes a new signaling pathway for SK channels by which RyRs modulate Ca2+ signaling in the heart.CaAdult C57B L mice were obtained from the Experimental Animal Center of Henan Province, China (No. SCXK-2010-0001). All of the animal care methods and procedures were approved by the Committee on the Ethics of Animal Experiments of the University of Zhengzhou (No. SYXK-2010-0001). This investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health.2 inhalation. The mice hearts were quickly removed and subjected to enzymatic digestion via Langendorff perfusion. Single atrial cells were isolated and stored in a high-K+ solution for 2 h at room temperature before the electrophysiological recordings.Single mouse atrial myocytes were isolated using a previously described enzymatic method 2+-activated K+ current  was recorded from freshly isolated atrial myocytes at room temperature using the previously described voltage-clamp protocol The whole-cell configuration of the patch-clamp technique was used. The Ca2+]i) transients were recorded on freshly isolated cardiomyocytes or transduced single myocytes previously loaded with the fluorescent Ca2+ dye Fluo-3AM . The samples were divided into five groups, including the control , ryanodine+caffeine, ryanodine+thapsigargin+caffeine, Lenti-GFP+caffeine and Lenti-siRyR2+caffeine. The control cells were dispersed in Tyrode's solution , and the other groups were dispersed in Tyrode's solution supplemented with the appropriate inhibitor before performing the experiment. All the cells were stimulated at 1 Hz by field stimulation applied by two parallel platinum electrodes to reach steady state. When an inhibitor was used, the cells were first perfused with the inhibitor alone followed by the addition of 5 mM caffeine in the continuous presence of the inhibitor. The imaging data were recorded 1–2 min following the caffeine applications. All the imaging data were recorded in the line-scanning mode along the long axis of the myocyte excited at 488 nm with a confocal laser scanning microscope  and analyzed by Olympus Fluoview viewer (Japan). The Ca2+ level was reported as the fluorescence (F) over the fluorescence min (F0), where F0 is the resting or diastolic fluo-3 fluorescence.The calcium imaging experiments were performed as described elsewhere GCCATTCCTACAGTGGTAT, location 864, CGTCCACATACTATTACTC, location 4767, AGGACACCATCAATCTGCT, location 6837 and CACAGCCTATCATCAACAA, location 10404) were selected based on the cDNA sequence of mouse RyR2 (accession number NM-023868.2). Four pairs of oligonucleotides encoding shRNAs and a negative control shRNA were designed and chemically synthesized by Invitrogen. These sequences were subcloned into the HIV-based psiHIV-U6 plasmid . Recombinant lentiviral vectors were packaged and amplified in 293T cells using the Lenti-Pac™ HIV Expression Packaging Kit (Clontech) according to the manufacturer's protocol. The supernatant of the cultured 293T cells containing lentiviral particles was collected following transfection for 48 h.Four optimal 19-mer target sequences .Twenty-four hour old neonatal mouse hearts were dissected and subjected to sequential digests with a trypsin/DNase II (Sigma) solution at 37°C. The supernatant was collected at 3–5 min intervals. The dispersed cells were pre-plated for 2 h in DMEM (GIBCO) with 10% fetal bovine serum . The unattached myocytes were plated in 35-mm dishes in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS, 1% penicillin-streptomycin , 0.1 mmol/L bromodeoxyuridine , and 20 µmol/L arabinosylcytosine  at 37°C in an incubator with a mixture of air and 5% CO5) were incubated in Annexin V binding buffer containing PE Annexin V and 7-amino-actinomycin D (7-AAD) at room temperature in the dark for 15 min. The flow cytometry analysis was performed using a FACS Calibur system with FACSDiva software . The counts are expressed as a percentage of the total number of cells counted.The NMCMs infected with lentiviral vectors were harvested with trypsin and washed with cold PBS. The infected cells were monitored by GFP fluorescence. A PE Annexin V Apoptosis Detection Kit I (BD Pharmingen) was used to identify the apoptotic efficiency of the infected cells. The cells in each sample . The isolated RNA was treated with DNase at 37°C. cDNA was synthesized from the total RNA samples by oligo(dT)-primed reverse transcription using a RevertAid™ First Strand cDNA Synthesis kit (Fermantas). The quantitative real-time polymerase chain reaction (PCR) products were detected using a SYBR Green qPCR Kit (TAKARA) by Bio-Rad MiniOption (USA). The PCR primers used to detect RyR2 and GAPDH were: RyR2 (251 bp), Forward: 5′-GAATTCATCATGGATACTCTACC-3′ Reverse: 5′-GTCATGCACATTATCTTCTGCAT-3′; GAPDH (150 bp), Forward: 5′-TGTGTCCGTCGTGGATCTGA-3′, Reverse: 5′-TTGCTGTTGAAGTCGCAGGAG-3′. The RyR2 and GAPDH primers were designed and synthesized by Shanghai Bioasia Company (China). The relative quantification of RyR2 mRNA was determined by normalization of the threshold cycle (Ct) of these genes to a housekeeping gene, GAPDH.−1): NaCl 113, KCl 4.7, KH2PO4 0.6, Na2HPO4 0.6, MgSO4 1.2, NaHCO3 12, KHCO3 10, HEPES 10, taurine 30, 5% FBS and 12.5 µM CaCl2 for 10 min at room temperature. After the myocytes were pelleted by gravity, they were resuspended with a stepwise increase in [Ca2+]i to 1 mM.Freshly isolated atrial myocytes were suspended in modified Tyrode's solution containing  at room temperature. The myocytes were collected and plated in a 60-mm laminin-coated dish in MEM containing 2.5% FBS and 1% PS. After 1 h of culture, the medium was switched to FBS-free MEM, and an appropriate titer of gene-carrying lentivirus was added to the culture medium. The cells were maintained at 37°C in a 2% CO2 incubator for 36–48 h. A recombinant lentivirus containing GFP alone was used as a control. GFP-positive cells were used for the electrophysiological recordings.The prepared cardiac myocytes were resuspended in MEM containing 1.2 mM CaThe protein samples were extracted from cultured adult cardiac myocytes transduced with lentivirus containing Lenti-siRyR2 for 48 h. Thirty-five microgram aliquots of the protein samples were separated by 5% gradient SDS-PAGE for RyR2 and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Following transfer, the membrane was blocked and incubated with anti-RyR2 antibody  at 4°C overnight, respectively. The membrane was incubated with a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (Thermo). The bands were detected by chemiluminescence (Thermo). Quantification of the signals was performed by densitometry .Reciprocal Co-IP was performed as previously reported ANOVA followed by paired or unpaired Student's t tests, as appropriate. The differences were considered significant when P<0.05 and are generally indicated by an asterisk (#).The data are expressed as the means±S.E.M. Differences between the groups were evaluated by one-way Further details of the experimental procedures are available in the online supplementary material.K,Ca current (apamin-sensitive current) was recorded from a single atrial myocyte from 9-11 animals for each group in the presence or absence of apamin  and ryanodine  resulted in a significant negation of a component of the inward and outward currents in the atrial myocytes compared with the control cells  or by the combination of 20 µM ryanodine and 2 µM thapsigargin (n = 9 cells). There were significant differences in the normalized amplitude of the Ca2+ signal among the ryanodine alone, ryanodine and thapsigargin and control groups (p<0.01). There was no significant difference between the ryanodine group and the ryanodine and thapsigargin group . Our data further suggest a functional role of RyR-mediated Ca2+ release evoked by the Ca2+ influx on the SK channel activation in the cardiac myocytes.To directly examine the functional role of RyR2 knockdown on cardiac myocytes, whole cell patch-clamp techniques were used to record the Iin vivo co-immunoprecipitation assay. The cell lysate from adult mice atria was isolated, immunoprecipitated with anti-SK2 antibodies, and immunoblotted with anti-RyR2 antibodies. As shown in 2+ release. The structural platform for the channels' crosstalk might be the association with junctional membrane complexes The above results suggest the functional interaction of the SK2 channel with RyR2, but little is known regarding whether RyR2 associates with RyR2 in native mouse cardiac tissues. We examined the association of endogenous SK2 and RyR2 proteins in adult mouse cardiac muscle using an 2+ release. Our results show that the inhibition of the RyR2 Ca2+ release channel and the inhibition of the SERCA suppressed the SK2 channel current in cardiac myocytes. Activation of RyRs by caffeine increased the apamin-sensitive currents in the cardiac cells. This report illustrates the role of the RyRs-induced Ca2+ release from the sarcoplasmic reticulum in SK2 channel activation in cardiomyocytes.We used isolated mouse atrial myocytes and cultured cardiac myocytes infected with lentiviral-mediated small interference RNA targeting RyR2 to investigate the functional communication between the SK2 channels and RyRs-sensitive Ca+ current, which is consistent with previous reports 2+]i and on the subcellular location of the SK channels 2+ channel-mediated Ca2+ current and initiate the release of Ca2+ from the sarcoplasmic reticulum + while decreasing the driving force for the Ca2+ current. A component of the mainly outward current reflected the changes in the intracellular Ca2+ concentration in the cardiac myocytes 2+]i concentrations on the Ca2+-activated potassium current 2+]i significantly increased the Ca2+-activated potassium current in single cardiac cells compared to the concentration of 100 nm. In the present study, caffeine increased the Ca2+-sensitivity of RyRs and Ca2+ release from SR by elevating the amplitude of the Ca2+-activated potassium current. The inhibition of RyR2 by blocking it or by knockdown of RyR2 mRNA suppressed the Ca2+-activated potassium current. These findings suggest that blocking RyR2 might lead to a diminished Ca2+ supply for SK channel activation. The dynamics of [Ca2+]i could result in a change in the sensitivity of SK channels to transient Ca2+ release events In the present study, we used electrophysiological techniques to determine that the SK2 channel current is a time-independent inward rectifier of K2+ entry through the voltage-gated Ca2+ channels activates the SK channels in cardiac cells. Ca2+ influx through the VDCCs stimulates the opening of RyR2 and subsequent Ca2+ release from the SR 2+ that occurs in cardiac cells is not known.A previous study demonstrated that the SK2 channels in cardiac muscle are mainly coupled to the Cav1.3 channel 2+-activated potassium current in the mouse atrial cells by a RyR2 blocker and a RyR2 activator. We found that the apamin-sensitive current in the mouse atrial cells was abolished by siRNA RyR2 knockdown. Second, we found that the intracellular Ca2+ transient contributed to our understanding of the dependence of SK channel activity on SR Ca2+ release. Third, we tested the interaction between SK2 and RyR2 in mouse cardiac muscle using co-immunoprecipitation. RyR2 did not interact directly with the SK2 channel, as suggested by the YTH screen, but both proteins were present in native cardiac tissue as members of the same immunoprecipitated complex. All these phenomena lead to the conclusion that ryanodine receptor-mediated Ca2+ release activates small-conductance Ca2+-dependent K+ channels in cardiac myocytes. The combination of the published report 2+ influx directly through voltage-gated Ca2+ channels or indirectly through ryanodine receptor-mediated Ca2+ release from SR intracellular stores.We summarize the three principal findings that support our conclusions. First, we demonstrated the functional modulation of the RyR2 channel on the Ca2+ release to the SK channels in the cardiac myocytes. Giant inside-out patches showed that the onset of the SK current commences within 1 ms after Ca2+ application, with time constants of the SK channels activation of 5–15 ms and the time constants of deactivation of 22–38 ms 2+ ions only, the SK current could be repeatedly activated for as long as the patches remained intact without changes in the activation kinetics 2+-activated K+ current remains active after a transient outward K+ current (Ito) is inactivated It was reported that the SK2 channels profile the cardiac action potential repolarization, especially during the late phase of the cardiac action potential 2+ release and altered intracellular Ca2+ load. During the initiation of contraction, the modulation of RyR2 on the SK2 channel in cardiac myocytes contributes significantly to the cardiac electrical-mechanical cycle. It is important to define the functional communication between the SK2 channel and RyR2 in the cardiac myocytes because this communication indicates a new signaling pathway for SK channels by RyRs-induced Ca2+ release and signaling in the heart.SK channels have been shown to mediate after hyperpolarizations Text S1Supplementary Methods.(DOC)Click here for additional data file.


The down regulation of the canonical ORAI1 activator STIM1 during lactation suggests that other known ORAI activators such as STIM2 and SPCA2 may be important during lactation.The entry of calcium ions into mammary gland epithelial cells is one of the least well-understood processes in the transport of calcium into milk during lactation. The store-operated calcium entry channel ORAI1, has been suggested as a potential mechanism for the entry of Ca2+ influx. Silencing of Orai1 abolished this enhancement of Ca2+ influx. Stim2 had a modest effect on Ca2+ influx in this in vitro model of lactation, whereas Stim1 and Spca2 silencing had no effect. Despite pronounced increases in Spca2 mRNA during lactation there was no change in the generation of the alternative splice product generated by Mist1, which increases during lactation.Differentiation of HC11 mammary gland epithelial cells was associated with enhanced basal Ca2+ influx and this is associated with enhancement of basal Ca2+ influx. This enhanced Ca2+ influx appears to occur through the calcium channel Orai1.These studies support the hypothesis that lactation is associated with a remodelling of Ca The rapid growth of the neonate, particularly the calcification of bones and teeth, places a high demand for Ca2+ in milk. Depending on the species, the concentration of total Ca2+ in milk ranges from 8 to 60 mM CYT in differentiated HC11 cells with Stim2 silencing also support this role. Other mechanisms of ORAI1 activation during lactation have also been proposed, such as calcium store independent activation by SPCA2 CYT. The slope of the curve was calculated based on points at 0 to 5 s post Ca2+ addition and were expressed as a percentage relative to the average readings for differentiated cells treated with non-targeting siRNA with maximum Orai1-mediated Ca2+ influx induced by 10 μM cyclopiazonic acid (CPA).Calcium assays were performed using a fluorometric imaging plate reader (FLIPRescribed . Cells wβ-casein (Mm00839664_m1), Orai1 (Mm00774349_m1), Spca2 (Mm01242899_m1), Spca2 exon 15–16 (Mm01242904_m1), Spca2 exon 26–27 (Mm01242916_m1), Mist1 (Mm00487695_m1), Stim1 (Mm00486423_m1), and Stim2 (Mm01223102_m1) were amplified using the TaqMan® gene expression assays, and the data normalized to Ppib (Mm00478295_m1) and Actb (Mm01205647_g1). Data were analyzed using the comparative Ct method as described previously [Total RNA was isolated using the RNeasy Plus mini kit (Qiagen) as per the manufacturer’s instructions, and mRNA was quantitated using real time RT-PCR and a 7500 real time PCR system (Applied Biosystems). Mouse mammary tissues were obtained as previously described . Mouse βeviously . All datAll experimental treatments were conducted as three wells per experimental plate in quadruplicate. Results for the slope are presented as standard error of the mean (SEM) and statistical comparisons were performed using a two-way RM ANOVA matching by rows with a Bonferroni multiple comparison post-test. Real-time RT-PCR results are presented as standard deviation (SD) and were analyzed for significance using a paired t-test. All statistical analyzes were conducted using Prism Graph Pad, Version 5.04, Berkeley, CA, and significance was demonstrated at P < 0.05 where appropriate.The authors declare that they have no competing interests.DR helped design and performed experiments and data analysis and wrote the paper. CS contributed to the writing of the paper and preparation of lactation samples. IA contributed to the writing and revision of the paper. SRT designed experiments and contributed to the data analysis and writing of the paper. GM designed experiments, and contributed to analysis of the data and wrote the paper. All authors read and approved the final manuscript.HC11 cells were treated with siRNA for Orai1  (A.), Stim1 (B.), Stim2 (C.) and Spca2 (D.). Total mRNA was isolated 24 h after siRNA treatment before real-time. RT-PCR for assessment of gene silencing. Data were normalised to Ppib and ActB mRNA, and are shown as relative fold expression to siNT treated HC11 cells .Click here for file


Scratching confluent HaCaT monolayers decreased the number of cells displaying repetitive Ca2+ oscillations as well as the frequency of their Ca2+-transients in cells close to the wounded area and initiated migration of the cells into the wound bed. In contrast, calyculin-A (CLA) and okadaic acid (OA), known cell permeable inhibitors of protein phosphatase-1 and 2A, increased the level of resting [Ca2+]i and suppressed cell migration and wound healing of HaCaT cells. Furthermore, neither CLA nor OA influenced how scratching affected Ca2+ oscillations. It is assumed that changes in and alterations of the phosphorylation level of Ca2+-transport and contractile proteins upon phosphatase inhibition mediates cell migration and wound healing.Changes in intracellular calcium concentration ([Ca Calcium ions underlying Ca2+ oscillations are released from the endoplasmic reticulum (ER) via inositol 1,4,5-trisphosphate receptors (IP3R) and ryanodine receptors (RyR), and often spread through the cytoplasm as a regenerative Ca2+ wave In mammalian cells changes in intracellular calcium concentration (i have been implicated to possess a significant role in the regulation of cell migration their interaction has not been studied in wound healing. During wound healing, keratinocytes initiate migration from the wound edge by extending lamellipodia into a fibronectin-rich provisional matrix, which was enhanced by protein-serine/threonine kinase inhibitors Albeit protein kinase and phosphatase enzymes together with the changes in [Ca2+]i2+-transients occurring during Ca-oscillations may exert a resensitization-desensitization control during substrate guided movements of keratinocytes. Similar results were obtained with human primary keratinocytes where the Ca2+ uptake via nicotinic acetilcholine (Ach) receptors caused the decrease of the migratory distance of the cells 2+]i in human neutrophils 2+ release and entry was enhanced by both CLA and tautomycin (TM) in parotid acini, while OA had no influence on the release but inhibited entry 2+-transients and cell shortenings in both control and isoproterenol-treated cardiac myocytes 2+]i and phosphorylation of key proteins (by activating kinases or inhibiting phosphatases) may be interrelated and their combined effect might mediate cell migration.In fish keratinocytes migration can be stopped with a burst increase of [Ca2+ oscillations were observed and analyzed in unscratched cells and cells next to the scratch. In the latter increased resting [Ca2+]i and decreased oscillations could be observed. Phosphatase inhibitors increased both the extent of spontaneous Ca2+ oscillations and the resting [Ca2+]i, while they decreased migration of the cells. These results suggest that protein phosphatases and oscillatory changes in [Ca2+]i have distinct roles in keratinocyte migration.In our experiments migration and proliferation of HaCaT keratinocytes in the presence and absence of phosphatase inhibitors (CLA and OA) were tested following a scratch of confluent cells. Spontaneous Ca2 atmosphere as described in our previous reports HaCaT keratinocytes 2+]i was monitored using Fura-2, as described in our earlier reports 2, 1.8 CaCl2, 11.8 Hepes-NaOH, 1 g/L glucose, pH 7.4, all from Sigma) for 30 min at room temperature. Cells were incubated with CLA (10 nM) for 1 hour, or with OA (50 nM) for 2 hours before the measurements, and during the experiments cells were bathed continuously in these phosphatase inhibitors. The scratch was made with a sterile 10 µl pipette tip in the confluent culture. Coverslips with Fura-2-loaded cells were placed on the stage of an inverted fluorescent microscope . The excitation wavelength was changed between 340 and 380 nm by a microcomputer-controlled dual-wavelength monochromator , whereas the emission was monitored at 510 nm using a photomultiplier at 10 Hz acquisition rate of the ratios. [Ca2+]i was calculated from the ratio of fluorescence intensities (R = F340/F380) using an in vivo calibration  as described Resting i transients as observed by confocal laser scanning microscopy. 2+]i  at different timepoints. Note that not only cells marked by arrows but others as well presented clear changes in fluorescence intensity.Confluent HaCaT keratinocyte cultures displayed slow and repetitive spontaneous [Ca2+]i. Similar observations were made in case of the application of CLA and OA. For comparison, 0).2+ oscillations we tested a number of chemical agents known to either block the IP3R  or RyR (low concentration of tetracaine) and the Ca2+ATPase of the endoplasmic reticulum (thapsigargin). To assess the underlying mechanisms responsible for the Ca3R as well, significantly reduced both parameters. Similar observation was made with 10 µM thapsigargin. These results indicate that IP3R mediated Ca2+ release plays an important role in the generation of spontaneous oscillations of HaCaT keratinocytes.Tetracaine in the concentration assumed to affect only RyR neither influenced the percentage of oscillating cells nor the frequency of oscillations. On the other hand, both Xestospongin C, and tetracaine in 1 mM, where it is known to inhibit the IPp<0.05) in scratched cultures near the scratch as compared to before scratching . In case of CLA or OA treated cultures this significant difference was also observable between the corresponding scratched and unscratched cultures . In other words, cells near the scratch were less likely to display calcium oscillations independent of the presence or absence of phosphatase inhibitors.In control cultures the percentage of oscillating cells  decreasevs unscratched CLA treated cultures, where, albeit the tendency was present, the significance was not reached. These observations clearly suggest that while neither CLA nor OA influenced the effect of scratching, they both interfered with the basic mechanism responsible for the generation of the spontaneous increases in [Ca2+]i.Note, on the other hand, that the ratio of oscillating cells was significantly higher in cells treated with phosphatase inhibitors both before and after the scratch. This was true for all cases except for the unscratched control −3 Hz, n = 27) than in the unscratched control  (p<0.05). The presence of the phosphatase inhibitors did not alter significantly the frequency of oscillations as measured before and after the scratch was made . Nevertheless the same tendency was present in untreated and in CLA and OA treated cells, namely, the frequency of calcium transients was less in cells next to the scratch as compared to cells from unscratched cultures. Furthermore, the presence of phosphatase inhibitors also raised the frequency of oscillations in cells from both unscratched and scratched cultures as compared to control cells, although these differences were not significant .Resting [Caamplitude and the Full Time at Half Maximum (FTHM) of the individual spontaneous Ca2+ transients were examined. The former was calculated as the change in fluorescence intensity (0ΔF/F) and is a measure of the change in [Ca2+]i, while the latter was determined as the duration time of the transients at the half of the amplitude, and correlates with the duration of the Ca2+ transients.Two basic characteristics, the 2+ transients were significantly higher in the presence of phosphatase inhibitors than in control cultures , while no such increase was observable analyzing the data of cells next to the scratch . Furthermore, except for the CLA treated cells, the difference between the amplitude values measured in non-scratched cultures and in cells near the scratch was not significant . This difference was not statistically significant in case of CLA treated cells . In unscratched cultures CLA treatment decreased, while OA treatment increased the FTHM values. In cells near the scratch, the CLA treatment decreased significantly, while OA treatment did not change the FTHM values as compared to the control .The influence of the scratching on both parameters was parallel in control cultures, i.e. the amplitude as well as the FTHM increased. After the treatment by phosphatase inhibitors the measured values of these parameters (except for CLA treatment on FTHM) were also augmented.th hour of the experiment. In contrast, CLA and OA treated cells were not able to cover the scratched area during the same time interval, and, furthermore, in case of OA treatment cells lost their extensions and their connections with their neighbors by the end of the experiment , however, were significantly higher in the scratched area. These observations suggest that the Ca2+ release processes in cells next to the scratch are less frequent but last longer and result in a greater change in [Ca2+]i as compared with untouched cells. Other cell types like Cajal Our present results show that in cells from scratched regions the frequency of Ca2+]i and the frequency of Ca2+-oscillations in cells of both unscratched and scratched areas, however, cells close to the scratch still exhibited fewer number of oscillations than the unscratched ones. This [Ca2+]i increasing effect of phosphatase inhibitors is in accordance with previous results suggesting that phosphatase inhibition may raise [Ca2+]i and Ca2+ entry via enhancing the phosphorylation level of proteins involved in Ca2+-transport such as phospholamban, ryanodine receptor and plasma membrane Ca2+ channel 2+-transients  were calculated to be higher after the treatment by CLA and OA (except for FTHM in case of CLA treatment). These parameters showed remarkable alteration after scratching on phosphatase inhibitors treated cell cultures. Comparing the values measured on non-scratched cultures to the parameters obtained in cells next to the scratch, a synergistic relationship between phosphatases and the scratch-induced changes in [Ca2+]i could be excluded. The magnitude of the scratch-induced [Ca2+]i elevation was not significantly potentiated either by CLA or by OA treatment. The changes in the examined parameters observed in control cultures after scratching were not amplified by the application of phosphatase inhibitors rather they were diminished or vanished, however the application of phosphatase inhibitors on non-scratched cells had similar effect on the measured parameters as the scratch did.On the other hand, enhancing the phosphorylation level of proteins by inhibition of Ser/Thr specific protein phosphatases with cell-permeable CLA and OA increased resting [Ca2+]i and the phosphorylation state of certain proteins may contribute to the proliferation and migration of the cells during the wound healing process. According to the current literature this issue is controversial. On the one hand a burst increase in [Ca2+]i was reported to stop directional movement of keratinocytes Still standing key question is how changes in [Ca2+-dependent phosphorylation of the 20 kDa light chain of myosin II (MLC20) and RhoA/Rho-kinase induced phosphorylation and inactivation of myosin phosphatase 2+ flux and upstream activation of the p38/MAPK 2+]i as well as promoting the phosphorylation of both MLC20 and the inhibitory phosphorylation of the myosin phosphatase regulatory subunit as shown earlier Movement of cells requires remodeling of the actin-cytoskeleton that includes formation of actin-myosin stress fibers via the Ca2+-oscillations in cells close to the scratch may help keratinocytes to move into the gap between the cells caused by scratching. On the other hand lowering [Ca2+]i may also be advantageous for the proliferation of keratinocytes since this process could be inhibited by increasing influx of Ca2+ into the cells With respect to these findings the decrease of CaVideo S1Migration of keratinocytes in control culture. Time-lapse video. Control cells started to cover the scratched area, and they totally filled it in by the 17th hour of the experiment.(MP4)Click here for additional data file.Video S2Migration of keratinocytes in ocadaic acid treated culture. Time-lapse video. OA treated cells were not able to cover the scratched area and, furthermore, these cells lost their extensions and their connections with their neighbors by the end of the experiment.(MP4)Click here for additional data file.Video S3Migration of keratinocytes in calyculin A treated culture. Time-lapse video. CLA treated cells were not able to cover the scratched area, however, morphological changes similar to the OA treated cells were not recognized on CLA treated cultures.(MP4)Click here for additional data file.


Mice lacking Anion Exchanger Isoform 3 (AE3), a major cardiac Cl−/HCO−3 exchanger, appear healthy, but loss of AE3 causes decompensation in a hypertrophic cardiomyopathy (HCM) model. Using intra-ventricular pressure analysis, in vivo pacing, and molecular studies we identified physiological and biochemical changes caused by loss of AE3 that may contribute to decompensation in HCM. AE3-null mice had normal cardiac contractility under basal conditions and after β-adrenergic stimulation, but pacing of hearts revealed that frequency-dependent inotropy was blunted, suggesting that AE3-mediated HCO−3 extrusion is required for a robust force-frequency response (FFR) during acute biomechanical stress in vivo. Modest changes in expression of proteins that affect Ca2+-handling were observed, but Ca2+-transient analysis of AE3-null myocytes showed normal twitch-amplitude and Ca2+-clearance. Phosphorylation and expression of several proteins implicated in HCM and FFR, including phospholamban (PLN), myosin binding protein C, and troponin I were not altered in hearts of paced AE3-null mice; however, phosphorylation of Akt, which plays a central role in mechanosensory signaling, was significantly higher in paced AE3-null hearts than in wild-type controls and phosphorylation of AMPK, which is affected by Akt and is involved in energy metabolism and some cases of HCM, was reduced. These data show loss of AE3 leads to impaired rate-dependent inotropy, appears to affect mechanical stress-responsive signaling, and reduces activation of AMPK, which may contribute to decompensation in heart failure.Cl Expression of individual genes was normalized to expression levels of Gapdh, which were determined as previously described . Ionoptix Ionwizard Analysis Software  was used for analysis.Ventricular myocytes were isolated from adult mouse hearts as described previously . Proteins were resolved on reducing SDS polyacrylamide gels, transferred to nitrocellulose or PVDF membranes and incubated with specific primary antibodies followed by corresponding secondary antibodies . Protein bands were visualized using the KPL LumiGlo chemiluminescent substrate system . The primary antibodies for SERCA2a, phospholamban (PLN), phospho-Ser16 and phospho-Thr17 of PLN, the NCX1 Na+/Ca2+ exchanger, the L-type Ca2+ channel α2 subunit, the cardiac ryanodine receptor (RyR2), and sarcomeric actin (as a loading control) were the same as used previously ; MyBP-C (sc-67353: Santa Cruz Biotechnology); phospho-Ser282 myosin binding protein-C (MyBP-C) ; total and phospho-Ser22/23 cardiac troponin I (TnI) ; total and phospho-Ser473 Akt ; total and phospho-Thr172 AMPK .Hearts were harvested from anesthetized mice (15 μl of 2.5% Avertin/g bodyweight) and total homogenates prepared as described previously . For group comparisons, a mixed factor analysis of variance with repeated measures on the second factor was used. Individual comparisons were performed using two-tailed Student's In a previous study we analyzed the effects of the AE3 null mutation using mice of a mixed 129Svj and Black Swiss background and found no significant differences in cardiovascular performance  in which pacing up to 750 bpm was attempted, AE3-null mice tended to be less able than WT mice to sustain higher heart rates. Thus, in subsequent experiments, pacing was limited to 550 bpm, and heart samples were collected for biochemical studies described in later sections.A common feature of heart failure is a reduction in the positive FFR .Pacing in WT mice led to an increase in +dP/dt, from 8402 ± 296 mm Hg/s at 350 bpm to 11700 ± 470 mm Hg/s at 550 bpm; the corresponding increase in mutant mice was from 8112 ± 534 at 350 bpm to 10066 ± 588 mm Hg/s at 550 bpm Figure . BecauseBlood pressure and left ventricular systolic pressure in FVB/N mice remain at a relatively constant level as heart rate increases , indicative of impaired relaxation, is observed  was reduced  and PLN phosphorylated on either Thr17 or Ser16 .In previous studies, Cas Figure . Neithert Figure  nor the t Figure  was altein vivo. Immunoblotting showed that phosphorylation of Thr17 was not significantly different in WT and AE3-null hearts under basal conditions   that are below those occurring −3 cotransport with a stoichiometry of either 1:2 or 1:3  that occur in response to loss of AE3; however, it could also affect Na+-loading.NBCe1 was shown previously to be upregulated in the retina of AE3-null mice  decreases Ca2+ currents  are strongly affected by pHi (Papalouka et al., −3 has been shown to cause a sharp increase in contractility in isolated hearts, with no increase in systolic Ca2+, indicating a critical role for HCO−3 homeostasis (Fülöp et al., − homeostasis, when the AE3 knockout was crossed with an NKCC1 Na+-K+-2Cl− cotransporter knockout, the double mutant exhibited impaired contractility; however, it appeared likely that this was due to impaired Ca2+ loading as NCX1-mediated Ca2+ extrusion was increased (Prasad et al., The mechanisms by which loss of AE3 might lead to increased biomechanical stress or affect sensing and responses to such stress are unclear. We suggested previously that in cardiac myocytes, AE3 may act in concert with a NaThe data clearly show that Akt signaling is enhanced in AE3-null hearts during pacing. This would be expected to improve the heart's response to biomechanical stress; however, if Akt were chronically activated, as seems likely given the heart rates that occur naturally in awake mice, it could also have detrimental effects (Chaanine and Hajjar, −/HCO−3 exchange leads to an impaired FFR, which is a clear physiological correlate of the predisposition to heart failure in the TM180 model of HCM (Al Moamen et al., 2+ transport and Ca2+ imaging of isolated myocytes argue against the hypothesis that primary perturbations of Ca2+ handling are responsible for the physiological deficit, although it is possible that modulatory effects of AE3 and NBCe1 activities on Ca2+ channels could perturb Ca2+ handling at the high frequencies occurring in vivo. There were no differences in the levels of total or phosphorylated MyBP-C and TnI, making it unlikely that altered activities or Ca2+ sensitivity of these myofibrillar proteins contribute to the deficit. The most striking change was an increase in phosphorylation of Akt and a decrease in phosphorylation of AMPK during pacing at elevated heart rates. AE3 has been localized to both sarcolemmal and intracellular membranes, and on the sarcolemma it appears to be concentrated at costameres (Alvarez et al., in vivo pacing. The mechanisms by which loss of AE3 leads to a deficient response to biomechanical stress and the downstream events that lead to decompensation in heart failure, possibly including impaired AMPK activation, will be investigated in future studies.In summary, our results show that the loss of AE3-mediated Cl2+ imaging experiments were performed by Nabeel J. Al Moamen; data were analyzed by Vikram Prasad, Gary E. Shull, John N. Lorenz, and Nabeel J. Al Moamen; and the manuscript was written by Vikram Prasad and Gary E. Shull.The experiments were conceived by Vikram Prasad, Gary E. Shull, and John N. Lorenz; the biochemical analyses were performed by Vikram Prasad; the analyses of cardiovascular function were performed by Valerie M. Lasko, Michelle L. Nieman, and John N. Lorenz; isolation of cardiomyocytes and 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.


Ischemic heart disease is a leading cause of mortality. To study this disease, ischemia/reperfusion (I/R) models are widely used to mimic the process of transient blockage and subsequent recovery of cardiac coronary blood supply. We aimed to determine whether the presence of the growth hormone secretagogues, ghrelin and hexarelin, would protect/improve the function of heart from I/R injury and to examine the underlying mechanisms.2+]i) transients and caffeine-releasable sarcoplasmic reticulum (SR) Ca2+ were measured. In addition, RT-PCR and Western blots were used to examine the expression level of GHS receptor type 1a (GHS-R1a), and phosphorylated phospholamban (p-PLB), respectively. Ghrelin and hexarelin pre- or post-treatments prevented the significant reduction in the cell shortening, [Ca2+]i transient amplitude and caffeine-releasable SR Ca2+ content after I/R through recovery of p-PLB. GHS-R1a antagonists, [D-Lys3]-GHRP-6 (200 nM) and BIM28163 (100 nM), completely blocked the effects of GHS on both cell shortening and [Ca2+]i transients.Isolated hearts from adult male mice underwent 20 min global ischemia and 30 min reperfusion using a Langendorff apparatus. Ghrelin (10 nM) or hexarelin (1 nM) was introduced into the perfusion system either 10 min before or after ischemia, termed pre- and post-treatments. In freshly isolated cardiomyocytes from these hearts, single cell shortening, intracellular calcium ([Ca2+ content) to allow the maintenance or recovery of normal cardiac contractility. These observations provide supporting evidence for the potential therapeutic application of ghrelin and hexarelin in patients with cardiac I/R injury.Through activation of GHS-R1a, ghrelin and hexarelin produced a positive inotropic effect on ischemic cardiomyocytes and protected them from I/R injury probably by protecting or recovering p-PLB (and therefore SR Ca Cardiac ischemia is one of the leading causes of mortality in the world. It is caused by a temporary interruption of blood flow in the arteries of the heart In vitro global and in vivo regional ischemia/reperfusion (I/R) models have been developed to examine experimentally cardiac ischemia and subsequent reperfusion of the ischemic heart. The in vivo regional I/R model mimics atherosclerosis by ligating the left anterior descending coronary artery. The global I/R model for in vitro study blocks all perfusion of the heart for a given period. The latter can be easily implemented and affects a larger area with a less variability among different regions 2+ ATPase (SERCA2a) and Na+-K+ ATPase activities. This results in an accumulation of intracellular Ca2+ ([Ca2+]i) and Na+ ([Na+]i) 2+]i. The combined effects of ROS and [Ca2+]i overload also favor the opening of the mitochondrial permeability transition pore (mPTP), which induces cardiomyocyte apoptosis and necrosis In the past 50 years, great progress has been made to clarify the metabolic changes that occur following I/R Ghrelin is a 28 amino acid peptide produced in the stomach and is an endogenous ligand of the growth hormone secretagogue (GHS) receptor type 1a (GHS-R1a) in vitro to I/R rat hearts was shown to reduce the infarct size in vivo prior to in vitro I/R injury showed an improvement in cardiac function Previous studies have confirmed the protective effects of GHS on whole heart function after I/R. Administration of ghrelin in vivo and in vitro I/R models have revealed some potential mechanisms of the cardioprotective effects of GHS. Detailed cellular and molecular pathways employed by GHS through activation of GHS-R1a after cardiac I/R remain elusive. Since [Ca2+]i plays a critical role in cardiomyocyte contraction and I/R injury, in this study we investigated the alterations in and regulation of [Ca2+]i homeostasis in isolated mouse cardiomyocytes with or without I/R and GHS treatment.As discussed above, whole heart functional studies employing the All experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health , and the protocol was approved by the Animal Ethics Committee of the University of Queensland (AEC # SBMS/814/07/NHMRC). All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.Human ghrelin was obtained from Auspep . Hexarelin was obtained from GL Biochem . Pentobarbital sodium was purchased from Virbac Pty Ltd . Heparin sodium salt was purchased from Sigma Aldrich . Fura 2-AM was purchased from Invitrogen . [D-Lys3]GHRP-6 was purchased from Anaspec Inc. . BIM28163 was kindly provided by Michael D. Culler . Other chemicals for recording solutions were purchased from Sigma .2, 10 Glucose, 20 Taurine, 1.5 CaCl2; pH 7.4; bubbled with 100% O2 at 37°C).Adult male C57/B1 mice (7 to 9 weeks old) weighing between 34 g and 36 g were anesthetized with sodium pentobarbitone  containing heparin . The heart was rapidly excised, cannulated and perfused retrogradely via the aorta with Tyrode solution at 3 ml/min on a Langendorff perfusion apparatus  or hexarelin (1 nM) was administered in the perfusion solution before or after ischemia for 10 min The times for stabilization, ischemia and reperfusion were similar to previous studies 2, 0.6 mg/ml collagenase Type II  and 0.1 mg/ml proteinase type XXIV . The Ca2+ level was gradually increased to 1.5 mM over 30 min. The yield of this isolation was usually around 60 – 70%. Only cardiomyocytes that were quiescent with a rod shape, sharp edges and clear striations were used in this investigation. At least 3 hearts were used in each group.Following perfusion, cardiomyocytes were isolated from the left ventricle of each heart with Tyrode solution containing 100 µM CaClSarcomere shortening was measured as previously described 2+-tolerant cardiomyocytes were loaded with 5 µM Fura-2 AM  for 10 min at room temperature. Cardiomyocytes were observed through a Nikon fluor ×40 oil immersion objective and positioned for recording of Fura-2 fluorescence signals. During field stimulation at 0.5 Hz, cytoplasmic Fura-2 was excited by an IonOptix Hyperswitch dual-excitation light source  at 340 and 380 nm and emitted light collected in a photomultiplier tube. [Ca2+]i concentration was inferred from the ratio (R) of the intensity of the emitted fluorescence signals. Amplitude, time-to-peak, time-to-90% decay, and rate of rise (dR/dt) of the derived [Ca2+]i transients were determined by IonWizard software.The isolated and Ca2+ content, cardiomyocytes with cytoplasmic Fura-2 were paced at least 15 times at 0.5 Hz and then stopped. About 30s later, 10 mM caffeine was added to induce SR Ca2+ release. The area under the caffeine-induced [Ca2+]i transient  and its amplitude were used as a reflection of the SR Ca2+ content 2+]i was also measured to estimate the Ca2+ clearance ability of Na+-Ca2+ exchanger (NCX).For estimation of SR CaTotal cellular RNA was extracted from left ventricle, septum and right ventricle of mouse hearts using a TRIzol Plus RNA Purification kit . Single-stranded cDNA was synthesized from 2 µg total RNA with an iScript cDNA Synthesis kit  following the manufacturer’s instructions.Taq DNA polymerase , the cDNA generated above and the corresponding primers for GHS-R1a TCATCGATCACAGCCATGT; Reverse: AAGCCAAACTGACCATGT; Tm  =  64°C, 40 cycles). Mouse 18s rRNA was amplified as a control. Following our previous report PCR was performed using JumpStart Protein expression of the GHS receptor GHS-R1a and the phosphorylated phospholamban (p-PLB)/phospholamban (PLB) that are essential for SERCA2a activity were examined by Western blot analysis according to previous reports post hoc test was carried out for multiple comparisons as appropriate. In all comparisons, the differences were considered to be statistically significant at a value of P < 0.05.All data were expressed as mean ± S.E.M. One-way ANOVA with Tukey The mRNA and protein expression of GHS-R1a, a GHS receptor in the mouse heart, was examined by RT-PCR and Western blots. As shown in 2+]i transients , which is consistent with previous reports describing increased cytoplasmic Ca2+ and possible Ca2+ overload after ischemia 2+]i after I/R was not reversed by either ghrelin pre-  and post-treatment , or hexarelin pre-  and post-treatment .Basal [Ca2+]i transients after I/R , Ca2+ release from SR through ryanodine receptors (RyR2s) or both.GHS treatment also influenced the time course of sarcomere shortening  and i transient amplitude  to activate RyR2s and thoroughly deplete the SR, resulting in a significant rise in [Ca2+]i . In addition, the prolonged time for maximal sarcomere shortening after I/R (2+ transients (2+]i transients after I/R (2+ current (ICaL) 2+ content  A previous study from our laboratory has found that ghrelin and hexarelin exert a positive inotropic effect on normally perfused rat hearts c effect  and 3. O2+ content  by GHS treatment, whereas the time-to-90% decay of [Ca2+]i transients under normal twitch had no significant change.For the first time, we have demonstrated that the positive inotropic effect of GHS is at least partially due to the recovery or maintenance of normal SERCA2a activity  and ther content . This is content  after GHl twitch  and the ansients , it sugg2+ overload, which has been reported as the main damaging factor causing cardiac dysfunction after ischemia 2+]i was increased in ischemic cells with or without GHS treatment. The basal level of [Ca2+]i would reflect the activity of both SERCA2a and RyR2s 2+ content was also restored. If the sensitivity of RyR2s to Ca2+ is changed in ischemia (Ca2+ leak from SR), increased Ca2+ leak from SR may occur. Such a presumption seems consistent with an even greater increase in the basal level of [Ca2+]i after GHS treatment. Moreover, the increased basal [Ca2+]i in GHS treated groups may also be due to the increased Ca2+ influx into the cell through ICaL,which has been reported previously by our group 2+ homeostasis and cardiomyocyte function seen in the current study requires further investigation.Ca2+]i transients of cardiomyocytes after I/R injury (2+]i transients, are mediated by GHS-R1a.We show here the expression of GHS-R1a in the mouse heart at both the mRNA and protein level . In ordeR injury . This suin vitro I/R injury and preserved the cell shortening by regulating [Ca2+]i. Both ghrelin and hexarelin prevented the decrease in the amplitude of [Ca2+]i transients and SR Ca2+ content after I/R by maintaining the ratio of p-PLB to total PLB and therefore the SERCA2a activity. Their positive inotropic effect on the cardiomyocytes and corresponding increase in [Ca2+]i transients is mainly mediated by the activation of GHS-R1a.In summary, ghrelin and hexarelin pre- and post-treatments protected mouse cardiomyocytes from


Tremendous progress towards the underlying molecular mechanism came with the discovery of the two respective limiting components, STIM and Orai. STIM1 represents the ER-located Ca2+ sensor and transmits the signal of store depletion to the plasma membrane. Here it couples to and activates Orai, the highly Ca2+-selective pore-forming subunit of Ca2+ release-activated Ca2+ channels. In this review, we focus on the molecular steps that these two proteins undergo from store-depletion to their coupling, the activation, and regulation of Ca2+ currents.Store-operated Ca Notably, although key regulatory cell processes are strongly dependent on the availability of elevated cytosolic Ca2+ levels, a cellular Ca2+ overload certainly entails the risk of cell death [Elevated cytosolic Canishment . Store-onishment , 84, 95.ed state , 81. In rization , 81. Howll death , 113.45Ca overlay experiments indicate this domain to form a Ca2+-binding pocket of low affinity [The molecular basis of CDI has emerged to be rather complex, as it involves not only the CRAC channel-forming proteins themselves but also depends on their respective binding stoichiometry. In the over-expression system, a high ratio of STIM1:Orai1 results in normal CDI characteristics, whereas lower STIM1 expression ratios reveal a reduced or even complete loss of CDI , 79. Thiaffinity . The exaaffinity  or it re2+ selectivity [All three Orai homologues exhibit CDI, yet differ significantly in the extent of inactivation. Orai3 currents inactivate most rapidly followed by Orai2 and Orai1 . The subectivity . A simil2+ influx due to the lack of CDI [2+ inward currents in store-depleted cells. Hence, this peptide may either interfere with relevant sites that are needed for proper channel function or can act as a blocking particle occluding the pore from the inside, thereby preventing ion conduction [On the cytosolic side, the second intracellular loop additionally modulates the fast inactivation process , 86. Subnduction . Considenduction . It is onduction . Hence, 2+-dependent inactivation [2+-dependent manner to a conserved region within Orai1 N terminus, thereby promoting CDI [Interestingly, in contrast to the reconstituted system, native CRAC currents come along with a much more pronounced Cativation , 113. Thtivation , 78, or ting CDI , 60. SubStore-operated activation is the most widespread way to stimulate the STIM/Orai channel pathway, however, alternative mechanism are starting to emerge .2+ influx independent of store depletion. Temperatures above 37 °C yield a STIM1 movement into junctions probably due to unfolding of the luminal EF-SAM domain that mimicked store-depleted conditions. Although heating has induced STIM1 clusters, functional coupling to Orai1 is inhibited. The high temperatures may enhance the STIM1 K-rich domain-mediated targeting of STIM1 and at the same time prevent or disrupt CAD domain-Orai1 interaction. Only after cooling below 37 °C, Orai1-mediated Ca2+ influx is observed, which therefore represents a temperature-dependent heat-off response of the cell. This heat response is unique to STIM1 and Orai1, and is not only observed in a heterologous expression but also for endogenous CRAC channels of Jurkat T cells. Therefore, it seems that STIM1 serves as a molecular temperature sensor in immune cells.Xiao et al.  have sho2+ level [ S-glutathionylation of Cys 56 independent of ER Ca2+ levels. This amino acid is located in the luminal part near the EF hand of STIM1. Its S-glutathionylation probably reduces the Ca2+ binding affinity of the nearby EF hand and consequently leads to STIM1 oligomerization and constitutive Ca2+ influx independent of store depletion. The effect of glutathionylation on STIM1 activation indicates that STIM1 is important in redox sensing [2+ synergistically modulate inflammatory reactions.In lymphocytes, treatment with oxidants induces an increased intracellular Ca2+ level . Hawkins2+ level  demonstr sensing . Hawkins sensing  postulat2+ influx [Not only hyperoxia affects STIM1 but also oxygen depletion, which leads to hypoxic stress and rapidly and reversibly attenuates STIM1/Orai1-mediated Ca+ influx . The rap+ influx .Mancarella et al.  propose 2+ channels have been investigated in detail over the last two decades, with the molecular key players of the CRAC channels, i.e., STIM1 and Orai1, identified in 2005 and 2006, respectively. This paved the way for thorough characterization of the molecular processes and structure–function relationships central to CRAC current activation, permeation, and inactivation. However, several processes within the STIM1/Orai-coupling machinery are less well understood, urgently awaiting 3D atomic resolution of Orai alone and in complex with STIM1 to provide further insight into their coupling. In particular, the molecular mechanisms as to how STIM1 oligomerization leads to CAD/SOAR exposure, the STIM1 domain(s) interacting with Orai N terminus and the transformation of this interaction into Orai gating are not yet clear.Store-operated Ca2+-activated adenylate cyclase (AC8) has been identified as an integral component of the STIM1/Orai1 signalplex [2+-signaling microdomain [Another field that has just opened comprises the identification of further proteins modulating or participating in the STIM1/Orai signalplex. Recently, SARAF , junctatgnalplex  further rodomain .Another topic related to STIM/Orai covers their physiological as well as pathophysiological role in various tissues and clearly shows an exponential rise in publications. Related to this, the development of drugs specific to the STIM/Orai system  though s


Chronic spontaneous urticaria is a common condition encountered in childhood, but long lasting persistent urticaria is less frequent. Autoimmune mechanisms may explain up to 30-50% of chronic idiopathic urticaria in adults, but such etiology has been less studied in children.Case report and literature review.We present the case of a 10 year old, otherwise healthy boy, who has a 6-7 year history of persistent chronic urticaria requiring ongoing treatment. His symptoms consist of daily urticarial lesions and angioedema with frequent flares. Upper respiratory viral infections, spring/ fall season and stressful events make his condition worse; however, no food or medication triggers has been found so far. The initial work up of chronic urticaria included CBC, differential, TSH, thyroid antibodies, ANA, C3 and C4 titers, serology for H. Pylori, tryptase level, ESR, stool for O&A, urinalysis, which were entirely negative. A year ago, he developed sudden onset of weight loss, polydipsia and polyuria, and was diagnosed with Type 1 insulin dependent diabetes. He was found to have positive antiGAD antibodies, but thyroid and anti TTG IgA antibodies remained negative. He has been tried on many treatment modalities including various combinations of new and old generation antihistamines, steroids, ketotifen, montelukast, but his condition remains active.As extension to current guideline for work up of chronic urticaria, besides thyroid and high affinity anti FcεRI receptor autoantibodies, screening for anti GAD and antiTTG IgA antibodies can be considered in cases of persistent long lasting chronic urticaria in childhood.


CLO-TOR was pro-apoptotic in an AML patient blast subset (CD34+/CD38−/CD123+), which is enriched in putative leukemia initiating cells (LICs). In summary, the CLO-TOR combination could represent a novel valuable treatment for AML patients, also in light of its efficacy against LICs.Signaling through the phosphatidylinositol 3-kinase (PI3K) pathway and its downstream effectors, Akt and mechanistic target of rapamycin (mTOR), is aberrantly activated in acute myeloid leukemia (AML) patients, where it contributes to leukemic cell proliferation, survival, and drug-resistance. Thus, inhibiting mTOR signaling in AML blasts could enhance their sensitivity to cytotoxic agents. Preclinical data also suggest that allosteric mTOR inhibition with rapamycin impaired leukemia initiating cells (LICs) function. In this study, we assessed the therapeutic potential of a combination consisting of temsirolimus [an allosteric mTOR complex 1 (mTORC1) inhibitor] with clofarabine, a nucleoside analogue with potent inhibitory effects on both ribonucleotide reductase and DNA polymerase. The drug combination (CLO-TOR) displayed synergistic cytotoxic effects against a panel of AML cell lines and primary cells from AML patients. Treatment with CLO-TOR induced a G Acute myeloid leukemia (AML) is defined as a clonal disorder characterized by the uncontrolled proliferation and survival of immature myeloid progenitors that undergo a differentiation block at various maturation steps, leading to accumulation of leukemic cells in bone marrow and inhibition of normal hematopoiesis . Despite©) in a group of elderly patients with refractory/relapsed AML . Cell lysates were clarified by centrifugation , and supernatants were incubated  with 7-Methyl-GTP-Sepharose beads  in 400 μL binding buffer . Beads were then washed 3 times in binding buffer and boiled in Laemmli's sample buffer [A total of 5 ×10−ΔΔCt.Total RNA was extracted using the RNeasy Mini Kit  according to the manufacturer's instructions and 1 μg of total RNA was reverse transcribed using Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) and Oligo(dT) 15 primer in the presence of dNTPs . STAT3 and c-Myc gene expression was assessed using The TaqMan® Gene Expression Master Mix, the assays Hs00374280_m1 and Hs00905030_m1 respectively, and the 7300 real-time PCR system . Results were normalized to the level of the ubiquitously expressed RNA 18S ribosomal 1 gene  and were expressed as 2The data are presented as mean values from three separate experiments ± s.d. Data were statistically analyzed by a Dunnet test after one-way analysis of variance (ANOVA) at a level of significance of p < 0.05 vs. control samples.