As activation of BK channels leads to hyperpolarization of the cell, these channels provide a negative feedback mechanism for Ca2+-induced functions. Many cellular functions of the retinal pigment epithelium (RPE) are coupled to changes in [Ca2+]i. The aim of this study was to identify which Ca2+-entry pathway leads to the activation of BK channels in the RPE.In different tissues the activation of large conductance Ca2+]i ARPE-19 cells were loaded with Fura-2.We used freshly isolated human RPE cells and the ARPE-19 cell line for the detection of transcripts of BK channel α subunits. Patch-Clamp measurements were used to characterize BK channels in ARPE-19 cells electrophysiologically. To monitor changes in [Ca2+]i by 34.16%. Nevertheless, caffeine-induced Ca2+ signals were not sufficient to activate BK channels. Instead, the activation of L-type Ca2+ channels by BayK 8644 caused a dramatic increase in BK channel activity and a shift of the reversal potential of the ARPE-19 cells by −22.6 mV.Freshly isolated human RPE cells and ARPE-19 cells were shown to express BK channels. In ARPE-19 cells these channels were shown to be functionally active. Application of iberiotoxin led to a block of outward currents by 28.15%. At +50 mV ARPE-19 cells had a BK channel-mediated current density of 2.42 pA/pF. Activation of ryanodine receptors by caffeine led to a significant increase in [Ca2+]i that are mediated by the opening of voltage gated L-type Ca2+ channels. As Ca2+ entering the RPE cells through these Ca2+ channels are known to be important for growth factor secretion and light-induced transepithelial transport, we speculate that BK channels coupled directly to these Ca2+ channels may provide a good tool for negative feedback control of the L-type Ca2+ channels.We have shown here for the first time that human RPE cells express BK channels. These channels are activated in RPE cells by increases in [Ca Accordingly, BK channels may serve as negative feedback regulators for events that depolarize the cell or lead to raises in [Ca2+]i. In neurons, they have been shown to control secretion of neurotransmitters in cooperation with voltage-gated Ca2+ channels i regulate a variety of cell functions: dark adaptation of photoreceptor activity, transepithelial transport, phagocytosis, growth factor secretion, and differentiation [2+]i, in turn, is controlled by different transporters and channels. Intracellular Ca2+ stores are depleted by activation of inositol 1,4,5-trisphosphate and ryanodine receptors i, they all potentially activate BK channels. Until now, BK channels in the RPE have been shown to be activated by oxidizing agents [2+]i [2+ source responsible for increases in [Ca2+]i that leads to the activation of BK channels in the RPE. The aim of this study was to identify which Ca2+ entry pathway leads to the activation of BK channels in the RPE.In the retinal pigment epithelium (RPE), changes in [Cantiation . [Ca2+]ieceptors . In the channels –11 and cchannels ,13) haveen shown . While T stimuli –19. As ag agents , by hypog agents , by mechg agents , and by ts [2+]i . But not2 and were passaged twice a week. For RNA isolation and Ca2+ measurements, confluent cultures of ARPE-19 cells were used up to passage number 75. For electrophysiological measurements, cells were passaged the day before the experiments were conducted to get isolated single cells.The human retinal pigment epithelial cell line ARPE-19  was cultured in Dulbecco’s modified eagle medium:F-12 nutrient mixture , which contained 10% fetal bovine serum (Invitrogen), 0.05% insulin-transferrin-sodium , 1% nonessential amino acids (Invitrogen), 100 U/ml penicillin and 0.1 mg/ml streptomycin (both Invitrogen). Cells were cultured at 37 °C in a humidified ambient atmosphere containing 5% CO2+ and Mg2+ (Invitrogen) to wash away residuals of the neuronal retina. A fine pair of forceps was used to gently brush away the RPE. The RPE cells were collected and lysed in the lysis buffer of the RNeasy Mini Kit . Total RNA from ARPE-19 cells was prepared from confluent cultures grown in a 25 cm2 culture flask. RNA was isolated using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. Next, 1 µg RNA was reverse transcribed at 37 °C for 1 h in the following reaction mixture: 1 µg oligo dT primer (Invitrogen), 1 mM of each dNTP, 20 U RNAguard , and 20 U Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Invitrogen). For control PCR reactions human total brain RNA  was reverse transcribed under the same conditions. PCR experiments were performed with 1 µl cDNA in 50 µl PCR reaction mixtures with Taq DNA polymerase (Stratagene). The following oligonucleotides specific for the BK channel gene kcnma1 (NM_002247) were used: forward GGA ATG GGA GAC GCT TCA TA, reverse CCT GCA GCG AAG TAT CAT CAT CA. For amplification, we used 40 cycles as follows 30 s at 95 °C, 30 s at 60 °C, and 60 s at 72 °C. The identity of the amplification product was confirmed with agarose (1.5%) gel electrophoresis with O'RangeRuler™ 100 bp DNA Ladder  and by sequencing using the Big Dye TerminatorTM kit . RT PCR experiments were repeated 3 times.Human RPE was obtained from organ donors within 24 h of death. The human donor eyes were obtained after corneal transplantation from the cornea tissue bank of the Eye Hospital at the University Hospital Hamburg-Eppendorf. For the use of human material, tenets of the Declaration of Helsinki were followed, informed consent was obtained from the relatives of the donors, and Institutional Human Experimentation Committee approval was granted for the studies. After the cornea was removed for transplantation, whole eyes without cornea were delivered ice-cold and processed for RPE preparation. The anterior parts of the eyes, including the vitreous and the retina, were removed. The posterior portion was rinsed with ice-cold D-PBS without Ca2, 2 mM CaCl2, 10 mM HEPES, 5 mM glucose, adjusted to pH 7.3 with NaOH. Patch-clamp electrodes with a resistance of 3–5 MΩ were pulled from borosilicate glass using a DMZ Universal Puller . The electrodes were filled with a solution containing 140 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 2.5 mM EGTA, 10 mM HEPES, adjusted to pH 7.3 with KOH. Whole-cell currents were measured using an EPC-9  patch-clamp amplifier in conjunction with TIDA software (Heka) for data acquisition and analysis. Fast and slow capacity transients were compensated. Series resistance errors were compensated to at least 75%. All experiments were performed at room temperature (22–25 °C).ARPE-19 cells were placed in a bath chamber on the stage of an Axiovert 35 inverted microscope . The bath solution was composed of 130 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1.8 mM CaCl2, 1.2 mM NaSO4, 2 mM NaH2PO4, 9 mM glucose, and 20 mM HEPES, adjusted to pH 7.4 with NaOH. Cells were then loaded with Fura-2 AM ester  for 40 min in the dark at room temperature in Ringer solution containing 10 µM Fura-2 AM Next, cells were washed and then incubated for at least 30 min with Krebs Henseleit solution. The coverslips were placed into a bath chamber perfused constantly with Krebs Henseleit solution and mounted onto a Zeiss inverted microscope (Axiovert 35) equipped with a 40X Fluar objective. Substances were applied extracellularly by the perfusion system. In control experiments [Ca2+]i was measured during changes between running and arrested perfusion. The absence of changes in [Ca2+]i during this maneuver indicated no influence of mechanosensitive channels when using the perfusion for drug application. Furthermore, in experiments which aim to investigate the role of cytosolic Ca2+ stores 50 mM caffeine was applied. Analysis of the cell size during caffeine application revealed no hyperosmotic shrinkage of cells: 100% before caffeine application; 99.87%±0.28%  at the end of caffeine application. We performed ratiometric measurements Fura-2 fluorescence at 5 s intervals using a high-speed polychromator system  altering the wavelength of excitation light between 340 and 380 nm. Emitted light was filtered with a 510 nm filter and detected by a cooled charged-coupled device camera . Data were collected with MetaFlour software  and analyzed with MetaAnalysis software (Molecular Devices). To calculate intracellular free Ca2+ ([Ca2+]i), we superfused cells with the Krebs Henseleit solution without Ca2+ supplemented with 1 mM EGTA and 1 µM ionomycin. The cells were then superfused with Krebs Henseleit solution supplemented with 1 µM ionomycin and saturating Ca2+ concentration. [Ca2+]i was calculated according to Grynkiewicz et al. [2+]=Kd *(R-Rmin)/(Rmax-R)*Sf/Sb. Where R is the fluorescence intensity ratio (F(340)/F(380)), Rmin the value where R is minimal (with EGTA), Rmax the value when R is maximal (with saturating Ca2+ and ionomycin). Kd is the dissociation constant of Fura-2 and Sf and Sb the maximal respectively minimal fluorescence after excitation with 380 nm.The ARPE-19 cells grown on coverslips to confluency were washed with Krebs Henseleit solution, which contained 118 mM NaCl, 5 mM KCl, 1.2 mM MgClz et al.  [Ca2+]=KHalf-maximal activation was obtained from a fit with a Boltzmann equation:1/2 is the potential of half-maximal activation and k is the slope factor. Activation time constants were calculated by fitting current traces by a mono-exponential function:where Vwhere τ is the time constant. Results were presented as mean ±SEM. Statistical significance was tested using one-way ANOVA. n=number of independent experiments, * stands for statistical significance with p<0.05.A typical recording of outwardly rectifying whole cell currents from ARPE-19 cells is shown in Although the presence of BK channels in RPE cells has been demonstrated in cultivated rabbit RPE cells and in another human RPE cell line (RPE-50), their expression has not yet been demonstrated at the mRNA or protein level. In 2+ released from intracellular Ca2+ stores through activation of ryanodine receptors leads to increased activity of BK channels. As the existence of ryanodine receptors in RPE cells has been postulated by binding studies using melanosomes isolated from RPE cells and 3H-labeled ryanodine [2+]i in RPE cells. We used 50 mM caffeine to stimulate ryanodine receptors. In 2+-response of a confluent ARPE-19 culture to caffeine stimulation is shown. Caffeine led to a significant increase of [Ca2+]i of 34.16±10.16% . After washout of caffeine the cytosolic free Ca2+ recovered to 83.3±6.2% of the basal Ca2+ level measured before application of caffeine . The initial increase in [Ca2+]i induced by application of caffeine should lead to an activation of BK channel, whereas the caffeine-dependent decrease in [Ca2+]i should lead to a deactivation of the BK channel. Thus, we investigated changes of membrane currents in ARPE-19 cells by patch-clamp measurements. Surprisingly, BK channels were not activated in response to the application of caffeine; instead, caffeine led to a clear downregulation of outward currents in RPE cells during the phase of reduced Ca2+ levels . However, the analysis of BK channel activity during caffeine application revealed that the initial rise did not increase BK channel activity whereas in the phase of reduced Ca2+ levels the activity of BK channels was subsequently reduced. Since the resting Ca2+ level results from the balance of Ca2+-ATPase activity and TRPC channels activity, it is likely that the basal activity of BK channels depends on the activity of TRPC channels [2+ channels by the specific agonist BayK 8644 did not lead to an general increase in [Ca2+]i. Consequently, with our setup for measurements of [Ca2+]i, we could not detect changes in [Ca2+]i, though we have shown previously that application of BayK 8644 leads to a strong activation of L-type Ca2+ currents i in the whole cell. Instead, the Ca2+ signal from one Ca2+ channel creates a nanodomain, and as a result, clusters of Ca2+ channels form microdomains [2+ signals strongly depend on the distance from the Ca2+ channel. At a distance of 200 nm they are already 10-fold smaller than at a 20 nm distance from the Ca2+ channel [2+]i that is only obtained in close vicinity to Ca2+ channels. Recently, a direct interaction between the α subunits of voltage-gated Ca2+ channels and BK channels has been demonstrated i –28. In schannels –33. The channels  and by tyanodine . We coul the RPE –11,18. Txchanger ,36. A vocurrents ,18,37. N studies ,39. Thisodomains . As a co channel . A changnstrated ,43. Actichannels ,43. HoweV1.3 subtype, which can show activation thresholds as negative as −40 mV i since it is maintained in inside-out patches exposed to different Ca2+ concentrations [The question arises whether functional L-type/BK channel interaction plays a role for RPE function. As mentioned in the previous section, native RPE cells express both L-type channels and BK channels. Thus, their functional interaction in native cells is likely. The next question would be how these ion channels may contribute to cellular functions, as they show activation thresholds which are far more positive than the resting potential of RPE cells of −50 mV. The L-type channels, which are expressed in RPE cells, are the channels of the Cas −40 mV . Furtherree Ca2+ . That BKCa2+ [2+ ,23. Whene values . In conte values  and Tao  the RPE –23. They the RPE , and act the RPE  and by s the RPE . The lattrations .2+ channels. Voltage-gated Ca2+ channels have been shown to be involved in different functions of the RPE. Long exposure of dark-adapted eyes to a light stimulus leads to slowly rising signal in the electrooculogram, the so-called light peak. This light peak is reduced by application of the L-type Ca2+ channel antagonist nimodipine, indicating that L-type Ca2+ channels are activated by light exposure [In this study, we provide data for an alternative mechanism for BK channel activation by possible direct coupling to voltage-gated Caexposure ,47. The 2+ channel-induced neurotransmitter release [2+ channels in RPE are responsible for Ca2+-induced secretion of vascular endothelial growth factor [In neurons, BK channels provide a negative feedback mechanism for Ca release . As L-tyh factor , it seem


We and others have reported a strong dose-dependent reduction in the incidence of prostate and lung cancer within populations exposed to boron (B) in drinking water and food; and in tumor and cell proliferation in animal and cell culture models.The global disparity in cancer incidence remains a major public health problem. We focused on prostate cancer since microscopic disease in men is common, but the incidence of clinical disease varies more than 100 fold worldwide. Ca2+ stores using cancer and non-cancer human prostate cell lines, Ca2+ indicators Rhod-2 AM and Indo-1 AM and confocal microscopy. In DU-145 cells, inhibition of Ca2+ release was apparent following treatment with Ringers containing RyR agonists cADPR, 4CmC or caffeine and respective levels of BA (50 µM),  or . Less aggressive LNCaP cancer cells required 20 µM BA and the non-tumor cell line PWR1E required 150 µM BA to significantly inhibit caffeine stimulated Ca2+ release. BA (10 µM) and the RyR antagonist dantroline (10 µM) were equivalent in their ability to inhibit ER Ca2+ loss. Flow cytometry and confocal microscopy analysis showed exposure of DU-145 cells to 50 µM BA for 1 hr decreased stored [Ca2+] by 32%.We examined the impact of B on Ca2+ release from ryanodine receptor sensitive stores. This occurred at BA concentrations present in blood of geographically disparate populations. Our results suggest higher BA blood levels lower the risk of prostate cancer by reducing intracellular Ca2+ signals and storage.We show B causes a dose dependent decrease of Ca CCE activates Ca2+ binding proteins that regulate numerous cell functions including gene transcription, cell proliferation, vesicle secretion, and apoptosis 2+ concentrations are returned to normal as Ca2+ is removed by transporters such as the Na2+- Ca2+ exchanger in the plasma membrane, the sarcoplasmic endoplasmic ATPase (SERCA) in the ER membrane, Ca2+ uniporter in mitochondria, and by binding to high affinity binding proteins 2+ release from RyR agonist sensitive sites.One of the ways cells respond to environmental stimuli is by opening channels between sites of stored calcium, such as the endoplasmic reticulum (ER), Golgi, and mitochondria (mt), which contain high free Cath most abundant element in seawater (425 µM B) and until recently dismissed as biologically irrelevant 3 boric acid and 1.6% as B(OH)4− borate. Biology has used this element in the structure of several molecules including: antibiotics in fungi Boron (B) is the 9Human blood levels of BA reflect the local geology, water quality, and plants in the diet with a world-wide range from 2 to 120 µM (21 to 1232 ng B/g wet blood) in free living healthy men and women + was reduced by phosphorylation and therefore potentially subject to biological regulation +/cADPR Ca2+ release pathway. We showed that pharmacological levels of BA (250 and 1000 µM), but not methylboronic acid, decreased NAD+ stimulated release of Ca2+ without affecting calcium release when applied on its own 2+ release by interfering with NAD+ conversion to cADPR, blocking release of Ca2+ stores, or interfering with CCE. It also raised the possibility that BA concentrations in the normal blood range might be able to inhibit Ca2+ release from ryanodine receptor (RyR) sensitive stores. Here we demonstrate that physiological levels of BA inhibit Ca2+ release from RyR responsive stores in human prostate epithelial cells and lower luminal Ca2+ levels.The biological plausibility for the chemopreventative effect of BA is supported by several lines of investigation. In immunocompromised mice, BA supplementation reduced the growth of transplanted human prostate tumors, decreased IGF-1 tissue concentrations, and lowered serum prostate specific antigen levels 2 humidified incubator. For confocal experiments, cells were cultured on glass cover slips for 24 hours prior to performing assays and studied at a confluency of less than 80%. DU-145 and PWR1E cells were acquired from American Type Culture Collection  and LNCaP was a gift from Dr Allen Pantuck of the Department of Urology, Geffen School of Medicine at the University of California, Los Angeles. RPMI cell culture media was supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), and L-glutamine (200 mM). PWR1E non-tumor cells were maintained in Keratinocyte media containing streptomycin (100 µg/ml), L-glutamine (200 mM), 2.5 µg human recombinant EGF, and 25 mg of bovine pituitary extract . Boron depleted media was prepared using the boron specific ionic exchange resin, Amberlite IRE-743 (Sigma) as previously described Experiments used prostate cancer cell lines DU-145 and LNCaP and the non-tumor cell line PWR1E. Cells were grown and maintained in cell culture plates at 37°C in 95% air and 5% CO2+ changes were monitored using the calcium sensitive dye Rhod-2, AM ester  which accumulates in organelles. Rhod-2, AM ester has a Kd of 1 mM and has been shown to compartmentalize, particularly in the mitochondria, but as we show, in other organelles as well  equipped with an Axoplan X63 (NA 0.95) water immersion objective. A HeNe laser was used to excite Rhod-2 at 543 nm. 488 nm from a laser diode was used to excite ER or Mt Tracker. The emission was collected on a photomultiplier tube through a 560 nm LP filter for Rhod-2 and a 505 LP filter for ER and Mito trackers. Additional magnification, time series, and background subtraction were controlled using Zeiss LSM acquisition software. All images were acquired as 12 bit.Storage Ca as well . We also tracker  [31]–st by 50 µM BA at 1 hour (2+ ]st (2+ release after DU-145 cells were pretreated with BA (50 µM) for 24 hrs and observed a 35% decrease in Ca2+ release , which increases cell growth, increased ER [Ca2+]ER, whereas TNF alpha (1 ng/ml), which reduces cell proliferation, reduced [Ca2+]ERThe importance of Ca2+ in a dose dependent manner and lower stored Ca2+ storage levels. This suggests that BA's ability to dampen Ca2+ signaling in cancer cells underlies its ability to reduce clinical cancer risk.In conclusion, BA has previously been reported to reduce the risk of prostate cancer in humans and reduce tumor growth and prostate cancer cell proliferation, migration, and invasion. Our results demonstrate that physiological levels of BA inhibit agonist stimulated release of stored Ca


The results of analysis of the dermatoglyphics of 152 children with acute lymphoblastic leukaemia (ALL) (and the first-degree relatives of 54 of them) contrast with those of 31 children with acute myeloblastic leukaemia (AML) (and the first-degree relatives of 25 of them). In ALL our findings suggest that neither genetic susceptibility nor an environmental factor, effective during the early antenatal period, is of aetiological importance; but the response to treatment, assessed as length of first remission, was found to be related to the amount of fingertip pattern. This may have clinical application. In AML there is evidence of a genetically determined factor carrying a high risk of the development of the disease, in that a member of each of 5 different families of the 25 studied bore a rare hypothenar pattern, compared with none in 75 control families. No dermatoglyphic features were of prognostic significance in AML.


Therefore, we designed a series of experiments to clarify the effects of actin filaments on mitochondrial Ca2+ storage, cytoplasmic Ca2+ concentration ([Ca2+]c), and the interaction between mitochondrial Ca2+ and cytoplasmic Ca2+ in Arabidopsis root hairs.Mitochondria are dynamic organelles that move along actin filaments, and serve as calcium stores in plant cells. The positioning and dynamics of mitochondria depend on membrane-cytoskeleton interactions, but it is not clear whether microfilament cytoskeleton has a direct effect on mitochondrial function and Ca2+ stores in the mitochondria of Arabidopsis root hairs. Simultaneously, these treatments induced an instantaneous increase of cytoplasmic Ca2+, followed by a continuous decrease. All of these effects were inhibited by pretreatment with cyclosporin A (Cs A), a representative blocker of the mitochondrial permeability transition pore (mPTP). Moreover, we found there was a Ca2+ concentration gradient in mitochondria from the tip to the base of the root hair, and this gradient could be disrupted by actin-acting drugs.In this study, we found that treatments with latrunculin B (Lat-B) and jasplakinolide (Jas), which depolymerize and polymerize actin filaments respectively, decreased membrane potential and Ca2+ release into the cytoplasm, and consequent changes in [Ca2+]c. We suggest that normal polymerization and depolymerization of actin filaments are essential for mitochondrial Ca2+ storage in root hairs.Based on these results, we concluded that the disruption of actin filaments caused by Lat-B or Jas promoted irreversible opening of the mPTP, resulting in mitochondrial Ca Rhizobia, and for pollen tubes responding to self-incompatibility factors c, is much lower than that outside the cell or in some inner membrane organelles [2+]c mainly depends on the cell wall or cellular Ca2+ stores, such as vacuoles, the endoplasmic reticulum (ER), and mitochondria c, which is assumed to be linked with and essential for root hair tip growth. The purpose of this study was to evaluate the effects of disruption of actin filaments on mitochondrial Ca2+ storage, [Ca2+]c, and the interaction between mitochondrial Ca2+ and cytoplasmic Ca2+ in root hairs.Tip-growing cells represent an ideal system in which to investigate the role of the actin cytoskeleton in signal transduction . Root ha2+ concentration. Both cable-like or dot structures dyes labeled were highly dynamic. In elongating root hairs, mitochondrial density was uneven and mitochondrial distribution showed a sub-tip to base gradient   Figure . The app2+ gradient . This result indicated that Ca2+ influx played a role in the [Ca2+]c elevation induced by Lat-B treatment.La+ Figure  or Ca2+-2+-ATPase, can increase cytoplasmic Ca2+ levels c increase in root hairs .Thapsigargin, a specific inhibitor of the ER Ca+ levels . In our s Figure . The sub2+]c, we used a scanning ion-selective electrode technique (SIET), to monitor Ca2+ fluxes through the root hair surface  from the ER  gradient from tip to base in living root hairs; and (3) actin-acting drugs first induced an increase followed by a decrease in [Ca2+]c. Based on these results, we conclude that the disruption of actin filaments induced the release of Ca2+ from mitochondria to the cytoplasm with an increase in [Ca2+]c; these increased cytoplasmic Ca2+ further induced the release of Ca2+ from the ER and caused changes in Ca2+ flux in Arabidopsis root hairs.In summary, our results indicate that disruption of actin filaments promoted an interaction between mitochondrial Cal Figure . Our datArabidopsis thaliana (Columbia 0) were surface sterilized, germinated on 1/2 MS medium supplemented with 0.1% (w/v) sucrose and 0.7% (w/v) plant agar, and grown at 22°C with a light/dark cycle of 16/8 h. The seedlings grew for 3-4 days and were chosen for experiments when roots were 1-2 cm long.Seeds of 35S::mGFP5-ER construct in transformed plants. Mitochondria and microfilaments were co-localized using MitoTracker and the 35S::GFP-FABD2 construct in transformed plants, respectively. After the plants were loaded with dyes, they were kept in dye-free medium for at least 30 min before starting the experiments.All dyes were from Invitrogen . Plants were incubated at 4°C in the dark for 30 min with one of the following dyes: 10 μM Rhod-2/AM (Rhod-2), 2 μg/ml JC-1, and 1 μM calcein/AM ; or for 1 h with 1 μM Fluo-4/AM, or for 15 min with 100 nM MitoTracker. The incubation with Fluo-4/AM was at 4°C and followed the method of Zhang . MitochoThe root hairs were viewed under a 63× water immersion objective. When scanning the fluorescence of mitochondria, we used Z-stacks scanning to detect the root hairs with normal streaming. Before treatment, the position of an individual slide was fixed and noted in the object stage, the buffer below the cover glass was absorbed with thin filter paper and the drugs were added gently with a 10 μL micropipettor. Images were collected before and after treatment respectively.Images obtained using a confocal laser microscope  were digitized at 8-bit resolution and analyzed using LSM 5 Image Browser or Image J software. Digital images were acquired every 5-10 s, and the average fluorescence intensities of all relevant regions were recorded and stored for subsequent analysis. Wide field fluorescence images were collected with exposure times of between 100 ms and 300 ms. The fluorescence of Fluo-4, Calcium green-1, calcein, and JC-1 were imaged using a 488 nm laser line for excitation, and the fluorescence was isolated using a dichroic mirror with mid-reflection at 505 nm and an emission filter (535 ± 15 nm). For Rhod-2, the emission was collected using a 535 nm primary dichroic mirror and the Meta-detector of the microscope.Arabidopsis plants, i.e. 6-8 root hairs selected randomly. Except for the analysis of density and concentration differences of the mitochondria from different regions of the root hairs, other data on mitochondria were obtained from the whole root hair rather than from certain specific region of the root hair. Before each experiment, root hair morphology and vitality were observed using transmitted light to ensure that normal cytoplasmic streaming was present in the growing root hairs. Each test was repeated at least three times for each root hair developmental period.The fluorescence patterns of Rhod-2 and JC-1 in root hairs showed punctuate structures representing individual mitochondria or their aggregates. Fluorescence intensity was measured in 1 μm-diameter regions of interest encircling the bright spots corresponding to mitochondria. The signals from 4-6 mitochondria within a cell were averaged. These observations were repeated 6-8 times in different 2+-sensitive fluorescent probes Fluo-4/AM ester and Calcium green-1-10-kDa dextran were used to measure [Ca2+]c in the root hairs. The latter Calcium green-1 was microinjected into the root hairs. Under an Axiovert 200 M inverted microscope , root hairs were impaled with micropipettes, pulled from filament electrode glass using a PC-84 puller , that contained 0.5 mM dextran-conjugated dye. The dye was then pressure-injected into the root hair base on the microscope. The injected cells were allowed to recover for 30 min prior to imaging after the microinjection.The Ca2+]m in the root hairs. [Ca2+] was calculated according to the following formula: [Ca2+] = Kd * (F - Fmin)/(Fmax - F), where F is the fluorescence of the indicator at the experimental Ca2+ levels, Fmin is the fluorescence in the absence of Ca2+ and Fmax is the fluorescence of the Ca2+-saturated probe. In the Fluo-4 and Calcium green-1 labeling experiments, each single measurement of the Fmax value was determined by addition of 2 μM ionomycin and 5 mM Ca2+, and the Fmin value was determined by addition of 5 mM EGTA, where the Kd of Fluo-4 was 345 nM and that of Calcium green-1 was 190 nM c: free cytoplasmic Ca2+ concentration; [Ca2+]m: free Ca2+ concentration in mitochondria; mPTP: mitochondrial permeability transition pore; F-actin: filamentous actin; G-actin: globular actin; ER: endoplasmic reticulum; Ψm: mitochondrial membrane potential; VDAC: voltage-dependent anion channel; ANT: adenine nucleotide translocase; CICR: Ca2+-induced Ca2+ release.The abbreviations used are: MF: microfilament; Lat-B: latrunculin B; Jas: jasplakinolide; Cs A: cyclosporin A; c gradient in a control root hairFigure S1. The [Ca. The root hair was micro-injected with Calcium green-1-dextran. Cytoplasmic calcium levels were pseudo-color-coded according to original intensity of green fluorescence. The bar on the right showed the relationship between Ca2+ concentration and cellular pseudo-color. Scale bar = 10 μm.Click here for file2+ levels induced by Lat-B in the root hairFigure S2. Changes in cytoplasmic Ca. (A) The pseudo-color image of the Ca2+ concentration labeled with Fluo-4 in a normally growing root hair. Scale bar = 10 μm. (B-J) The pseudo-color images of the same root hair treated with 500 nM Lat-B in 360 s duration. The bar on the right showed the relationship between Ca2+ concentration and cellular pseudo-color.Click here for fileFigure S3. Bright field image of illustrating the location of the scanning ion-selective electrodes in root hairs. In the experiment of Ca2+ flux measurement, the Ca2+ selective probe positioned 2 μm from the tip of root hair surface to record the plasma membrane Ca2+ fluxes. Scale bar = 10 μm.Click here for file


However, recent advances in muscle physiology research technologies have enabled the identification of other factors involved in length-dependent activation. The striated muscle sarcomere contains a third filament system composed of the giant elastic protein titin, which is responsible for most passive stiffness in the physiological sarcomere length range. Recent studies have revealed a significant coupling of active and passive forces in cardiac muscle, where titin-based passive force promotes cross-bridge recruitment, resulting in greater active force production in response to stretch. More currently, the focus has been placed on the troponin-based “on-off” switching of the thin filament state in the regulation of length-dependent activation. In this review, we discuss how myocardial length-dependent activation is coordinately regulated by sarcomere proteins.The basis of the Frank-Starling mechanism of the heart is the intrinsic ability of cardiac muscle to produce greater active force in response to stretch, a phenomenon known as length-dependent activation. A feedback mechanism transmitted from cross-bridge formation to troponin C to enhance Ca About a century ago, Otto Frank and Earnest Starling demonstrated a fundamental principle in cardiac physiology that an increase in ventricular volume enhances the systolic performance of the heart  , resulting in the binding of Ca2+ to troponin C (TnC) and the subsequent formation of cross-bridges . Unlike in skeletal muscle, cardiac myofilaments are not normally fully activated because the intracellular Ca2+ concentration ([Ca2+]i) is maintained relatively low (~10-6 M) in systole [3]. For relaxation to occur, four transport systems are involved to lower [Ca2+]i: (1) sequestration into the SR by the Ca2+-ATPase pump , (2) efflux via the sarcolemmal Na+/Ca2+ exchanger, (3) extrusion by the sarcolemmal Ca2+-ATPase pump, and (4) uptake into mitochondria via the Ca2+ uniporter. Myofilament relaxation takes place following dissociation of Ca2+ from TnC.First, we would like to summarize the cellular mechanisms of myocardial contraction and relaxation before discussing length-dependent activation. In cardiac muscle, contraction is regulated by micromolar concentrations of Cae system -5. Upon 2+-sensitive photoprotein aequorin and simultaneously measured [Ca2+]i and twitch force in living cardiac muscle. It was demonstrated that the rapid first phase occurs independently of [Ca2+]i, and the second phase is coupled with a rise of [Ca2+]i . It is now recognized that the former is controlled by myofilaments and it is responsible for the beat-to-beat basis modulation of the mechanical response to stretch, predominantly underlying the Frank-Starling mechanism. For the slow effect, the following mechanisms have been proposed. First, Ruknudin et al. [7] reported that stretch-activated channels capable of generating inward currents (including Ca2+) may operate to increase [Ca2+]i. Second, Alvarez et al. [8] provided evidence for stretch-induced activation of the sarcolemmal Na+/H+ exchanger by local autocrine/paracrine systems involving angiotensin II and endothelin-1, resulting in an increase in [Na+]i and a subsequent rise of [Ca2+]i (reviewed in [9]). Third, Petroff et al. [10] demonstrated that stretch induces PtdIns-3-OH kinase-dependent phosphorylation of both Akt and the endothelial isoform of nitric oxide synthase, nitric oxide production and an increase in Ca2+-spark frequency. Therefore, the stretch-induced production of nitric oxide may enhance the release of Ca2+ from the SR. In this article, we will focus on the rapid component of the force response after stretch, and discuss how this is integrated in the supramolecular structure sarcomere.When stretched under physiologic conditions, mammalian cardiac muscle exhibits a biphasic force response to stretch, in that twitch force increases immediately after stretch, followed by a slow and gradual increase in the force. In a pioneering study, Allen and Kurihara [6] used the CaLmax (where twitch force reaches the maximum) to ~90% Lmax at various points of isometric twitch contraction causes a transient increase in [Ca2+]i . Subsequent studies revealed that extra-Ca2+ is in proportion to the magnitude of force reduction [11], and is indeed markedly diminished in the presence of the actomyosin inhibitor 2, 3-butanedione monoxime (BDM) [12]. It is therefore considered that cross-bridge formation (or dissociation) enhances (or reduces) Ca2+-binding to TnC via a feedback mechanism, and both complementarily affect each other during twitch contractions. However, recent studies suggest that this is not the sole mechanism involved in length-dependent activation.Another important finding by Allen and Kurihara [6] is that a quick release of muscle length from 2+]) curve . This property has been confirmed by a number of researchers, and is now widely recognized to underlie length-dependent activation in cardiac muscle under physiologic conditions.The first direct evidence that myofibrillar contractility is increased with length in cardiac muscle was provided by Hibberd and Jewell [13] in experiments with chemically skinned preparations. They observed that active force increases with sarcomere length at submaximal levels, as well as at maximal levels, resulting in the leftward shift of the force-pCa  length-dependent activation in skeletal muscle is unaltered after TnC is exchanged for the cardiac isoform [16], and (2) overexpression of skeletal TnC in cardiomyocytes does not alter the length-dependent activation [17]. Also, the findings that lengthening increases (1) maximal Ca2+-activated force  and (2) Ca2+-independent, rigor cross-bridge-dependent active force  render this proposal further unlikely.A straightforward interpretation that can seemingly account for the findings by Hibberd and Jewell [13] may be that TnC plays a role as a “length sensor” that changes its affinity for CaInstead, it has been proposed that the leftward shift of the force-pCa curve results from an increase in the fraction of cross-bridges coupled with structural changes in the sarcomere. It is now widely accepted that the actomyosin interaction is a stochastic process . It is therefore considered that the attachment of myosin heads to the thin filament is enhanced as the distance between the thick and thin filament is reduced, as simulated by Ishiwata and colleagues based on a mathematical model . Indeed, osmotic compression reduces the lattice spacing and increases contractile force , coupled with enhanced cross-bridge formation [28]. Also, studies with synchrotron X-ray have provided direct evidence that the lattice spacing is reduced in response to stretch within the physiological sarcomere length range  in various animal species . These findings are consistent with the notion that the lattice spacing plays a pivotal role in length-dependent activation .1). N2BA titin contains an additional middle Ig segment, the N2A segment, and the PEVK segment of variable lengths. Therefore, its passive force is lower than that of N2B titin. The expression level of N2B and N2BA titins varies in a species-specific and location-specific manner, but both isoforms are expressed at similar levels in the ventricle of most mammals, including humans .What triggers lattice spacing reduction in cardiac muscle at the stretched state? Does it occur intrinsically in the sarcomere or with a support from the extracellular component such as the collagen network (as suggested in [19])? Recent studies have revealed that the giant sarcomere protein titin  is involved in the modulation of the lattice spacing . Titin is a muscle-specific elastic protein (3–4 MDa) that spans from the Z-line to the M-line in half-sarcomere . Titin develops passive force when stretched by external force, resulting from extension of its spring element in the I-band region of the sarcomere . While titin’s A-band portion is composed of relatively simple patterns of immunoglobulin (Ig)-like and fibronectin type 3 (Fn3) repeats, the I-band region has a complex sequence with distinct extensible segments: the tandem Ig segments (tandemly arranged Ig-like domains), the PEVK segment  and the segment that has a unique amino acid sequence (N2A or N2B). In cardiac muscle, two types of titin exist, i.e., stiff N2B titin (containing the N2B segment) and less stiff N2BA titin (containing both N2B and N2A segments)  in the sarcomere, resulting in the production of radial force as well as longitudinal force in the lattice; the former force may pull the thin filament closer to the thick filament . This likely increases the likelihood of myosin binding to actin. In the myofilament lattice, the electrostatic force (Fes) arising from the negative charge carried by thick and thin filaments tends to separate these filaments. Cazorla et al. [42] calculated the values of Fes at various sarcomere lengths and concluded that titin-based radial force and Fes are of similar magnitude in the cardiac myofilament lattice, at least in relaxing conditions. It is therefore likely that titin develops radial force that is large enough for it to play a role in counteracting Fes. Secondly, Fukuda et al. [19] applied limited trypsin treatment to rat ventricular trabeculae to selectively degrade titin, and found that the slope of the length-force curve became less steep. Since attenuation of the length-dependence was observed under conditions where the lattice spacing (as indexed by muscle width) was not significantly changed, it was concluded that a mechanism operating independent of the lattice spacing modulation was responsible. It has been reported that titin is an integral component of the thick filament, tightly binding to myosin and myosin-binding protein C [45]. Therefore, sarcomere extension by external force may cause mechanical strain of the thick filament via titin’s longitudinal force, allowing myosin to attach to actin. Indeed, Wakabayashi et al. [46] reported that in frog skeletal muscle, sarcomere extension causes an increase in the myosin periodicity, accompanied by a loss of characteristic resting order of myosin heads around the thick filament backbone. Thus, a similar mechanism may operate in cardiac muscle within the physiological sarcomere length, based on the higher level of stiffness compared to skeletal muscle. It is therefore reasonable to consider that titin modulates length-dependent activation and that the mechanism includes titin-based modulation of the lattice spacing and the thick filament structure .Two articles published in 2001 have significantly expanded our understanding of length-dependent activation by providing evidence that titin is involved in length-dependent activation. First, Cazorla β-Adrenergic stimulation induces activation of protein kinase A (PKA), resulting in the phosphorylation of various proteins in cardiomyocytes, causing marked alteration in systolic and diastolic performances of the heart (see [3]). It has been reported that PKA phosphorylates the N2B sequence of titin (both N2B and N2BA isoforms), resulting in a decrease in passive force, especially in the physiological sarcomere length range . Therefore, it may be possible that length-dependent activation is attenuated by PKA, as pointed out elsewhere [22]. 2+ sensitivity of force are not well correlated [50]. Therefore, factors other than the lattice spacing are likely involved in the regulation of length-dependent activation, obtained at the steady-state. Indeed, length-dependent activation reportedly varies as a function of recruitable  cross-bridges that have ATP and can, therefore, potentially produce force upon attachment to the thin filament . This notion is supported by the experimental evidence from different groups showing that the length dependency is attenuated at high cooperative activation states with a non-force-generating analogue of myosin subfragment 1 (NEM-S1) [51] or MgADP [18] and it is enhanced at low cooperative activation states with inorganic phosphate or H+ [20]. Therefore, the “on-off” equilibrium of the thin filament state, which is regulated by Tn under physiologic conditions, likely determines the magnitude of length-dependent activation in concert with titin-based regulation. However, the group of de Tombe challenged the lattice spacing hypothesis, providing evidence that the lattice spacing and Ca2+ receptor), TnI (an inhibitor of actomyosin interaction that tightly binds to either actin or Ca2+-TnC) and TnT  . Two metal binding sites exist in the C-terminal domain of TnC that bind both Mg2+ and Ca2+ with relatively high affinity. However, due to the higher concentration of Mg2+ under physiologic conditions (~1 mM compared with ~0.1 – ~1 μM for Ca2+), these sites are normally occupied by Mg2+, during both diastole and systole. Cardiac  TnC has one (two) regulatory Ca2+-binding site(s) with relatively low affinity in the N-terminal domain of this molecule. When [Ca2+]i is increased during systole, Ca2+ binds to the regulatory Ca2+-binding site, resulting in the onset of the conformational change of the thin filament. During diastole, the C-terminal domain of TnI tightly binds to actin, and tropomyosin blocks the actomyosin interaction (“off” state). However, when Ca2+ binds to the regulatory Ca2+-binding site of TnC during systole, the C-terminal domain of TnI is dissociated from actin and binds to the N-terminal domain of TnC, due to the enhanced TnC-TnI interaction (“on” state). It is generally considered that the transition from the “off” to “on” state is associated with a movement and rotation of tropomyosin on the thin filament . Previous studies have demonstrated that the “on-off” equilibrium depends on the isoform of Tn subunits , as well as on the fraction on the strongly bound cross-bridges, such as the actomyosin-ADP complex [58]. Tn is a heterotrimer of distinct gene products: i.e., TnC  attenuated the length dependency in skinned porcine ventricular muscle, to a magnitude similar to that observed in rabbit fast skeletal muscle . Considering the finding that Ca2+ sensitivity of force is increased in association with enhanced cross-bridge formation upon replacement with rabbit fast skeletal Tn [61], the switching of Tn from the cardiac to fast skeletal isoform likely accelerates the transition from the “off” state to the “on” state of the thin filament (similar to the finding in [60]). It is therefore considered that length-dependent activation is attenuated as a result of a decrease in the fraction of recruitable cross-bridges. Also, Terui et al. [61] demonstrated that an increase in titin-based passive force, induced by manipulating the pre-history of stretch (as in [30]), enhanced length-dependent activation, in both control and sTn-reconstituted cardiac muscles, suggesting that this phenomenon is coupled with both titin-based passive force and “on-off” switching of the thin filament state, with titin-based passive force acting as a trigger in this phenomenon.A mouse model expressing slow skeletal TnI  in the ventricle has been widely used to investigate the physiological properties of TnI under various conditions . Ventricular muscle expressing ssTnI shows greater Cavia a rise in [Ca2+]i. A simultaneous measurement of [Ca2+]i and twitch force in intact cardiac muscle revealed a tight coupling between Ca2+-binding to TnC and cross-bridge formation, both complimentarily affecting each other. Recent studies with skinned myocardial preparations demonstrated that at the steady-state, length-dependent activation is primarily regulated via “on-off” switching of the thin filament state in concert with titin-based lattice spacing modulation. Therefore, under physiologic conditions, cross-bridges recruited via this mechanism may enhance Ca2+-binding to TnC and consequently recruit more cross-bridges. Future work is warranted to fully elucidate the enigmatic phenomenon of length-dependent activation.In conclusion, myocardial length-dependent activation is the basis for the Frank-Starling mechanism of the heart, which is modulated by sarcomere proteins, rather than


Cerebral cortex is organized in layers which are characterized by distinct cytoarchitecture. We asked if astrocyte-dominant layer 1 (L1) of the somatosensory cortex was different from layer 2/3 (L2/3) in spontaneous astrocytic Ca2+ activity and if it was influenced by background neural activity. Using a two-photon laser scanning microscope, we compared spontaneous Ca2+ activity of astrocytic somata and processes in L1 and L2/3 of anesthetized mature rat somatosensory cortex. We also assessed the contribution of background neural activity to the spontaneous astrocytic Ca2+ dynamics by investigating two distinct EEG states (“synchronized” vs. “de-synchronized” states). We found that astrocytes in L1 had nearly twice higher Ca2+ activity than L2/3. Furthermore, Ca2+ fluctuations of processes within an astrocyte were independent in L1 while those in L2/3 were synchronous. Pharmacological blockades of metabotropic receptors for glutamate, ATP, and acetylcholine, as well as suppression of action potentials did not have a significant effect on the spontaneous somatic Ca2+ activity. These results suggest that spontaneous astrocytic Ca2+ surges occurred in large part intrinsically, rather than neural activity-driven. Our findings propose a new functional segregation of layer 1 and 2/3 that is defined by autonomous astrocytic activity.Cumulative evidence supports bidirectional interactions between astrocytes and neurons, suggesting glial involvement of neuronal information processing in the brain. Cytosolic calcium (Ca Astrocytes occupy a significant proportion of the cellular composition in the brain. Their functions have been considered to be logistic support for neurons, such as mediating energy metabolism for neurons or maintenance of extracellular medium concentrations 2+) level as neurotransmitters induce Ca2+ surge in astrocytes and many forms of gliotransmission are accompanied by cytosolic Ca2+ increase 2+ surges are reported: (1) neural activity driven Ca2+ surges, and (2) neural activity independent “intrinsic” Ca2+ surges. The neural activity driven astrocytic Ca2+ surge is triggered by activation of metabotropic neurotransmitter receptors, which leads to production of inositol 1,4,5-triphosphate (IP3), and thereby to release of Ca2+ from the endoplasmic reticulum (ER) 2+ surge is not known, but it is also mediated by IP3 receptor of ER 2+ activity is accumulating in in vitro experiments, an oft-expressed concern is the modification of cellular properties of astrocytes due to the invasive nature of the preparations Although astrocytes lack fast sodium action potentials, astrocytic activity can be monitored by cytosolic calcium (Cain vivo imaging studies have reported astrocytic Ca2+ surges upon optimized sensory stimulation 2+ surges in juvenile rats (<P16) Anatomically, neurons are organized in layers with characteristic cytoarchitecture in the intact cerebral cortex. Notably, with regard to cell bodies, L1 contains mostly astrocytes, while L2/3 is dominated with neuronal soma 2+ dynamics (1) reflected cytoarchitectural differences of L1 and L2/3 and (2) changed during two distinct EEG states. We found spontaneous Ca2+ surges of astrocyte somata in cortical L1 were nearly twice more frequent than that of L2/3. Furthermore, Ca2+ dynamics of processes within an astrocyte were independent in L1 while those in L2/3 were synchronous. EEG state comparisons, as well as pharmacological experiments showed insensitivity of the spontaneous Ca2+ surges of astrocytes to neuronal activities, suggesting that the Ca2+ surges were in large part “intrinsic”, rather than neural activity driven. Our findings propose a new functional segregation of layer 1 and 2/3 that is defined by autonomous activity of astrocytes.We asked whether spontaneous astrocytic Ca2+ sensitive fluorescent dye Oregon Green 488 BAPTA-1 (OGB-1) and the astrocyte marker Sulforhodamine 101 (SR101) was performed in the primary somatosensory cortex of adult (>P28) male Sprague-Dawley rats. In agreement with previously published literature, both neurons and glial cells were loaded with OGB-1 and cells with astrocytic morphology were labeled with SR101  and L1 astrocytes were significantly more numerous than that of L2/3  in L1 and 94 (22.2%) in L2/3. The average number of active astrocytes was 10.0±5.6 per imaging field in L1 and was significantly higher than 2.8±2.4 in L2/3  (2+ events per astrocytes (active and inactive) in thirty minutes was calculated to be 1.5±2.3 for L1 and 0.4±1.1 for L2/3 and the difference was significant . Among active astrocytes, L1 had 3.0±2.5 Ca2+ surges in thirty minutes and was significantly higher than L2/3 which had 2.0±1.7 Ca2+ surges . These astrocytic spontaneous Ca2+ events had of an average duration  than L2/3 (122.4±0.3)  totaled in 38 session for L1 and 34 sessions for L2/3 (see  t-test) . In orde t-test) . The meation see . The dur2+ surges, we calculated the histogram representing the frequency of Ca2+ surges of an astrocyte per thirty minutes for each layer (2 expected’). The Poisson distribution reflects unbiased random occurrences of Ca2+ surges using the mean counts of astrocytic Ca2+ events measured in the experiments . The experimental distribution deviated significantly from the Poisson distribution  were calculated   was applied on the cortical surface for >30 min before Ca2+ imaging. TTX had no noticeable effect on the ratio of astrocytes with spontaneous Ca2+ surges  and multi-unit activity was measured in the vicinity of the imaged area. The local field potential elicited alternating epochs of synchronized activity and desynchronized activity under urethane anesthesia . In the + surges . In L1,  = 0.35) . In L2/3 = 0.35) . These d2+ surges of astrocytes are due to action potential independent release of neurotransmitter or gliotransmitter such as glutamate and ATP, pharmacological experiments with antagonists were performed. As a result, we found neither MPEP , or PPADS  had no noticeable effect on percentage of astrocytes with spontaneous Ca2+ surges  were placed at each of the branches, one being more proximal (p), the other more distal (d) to the soma . The correlation coefficient was calculated for pairs of ROIs , in the case of L2/3, Ca2+ traces of ROIs placed at different processes of astrocyte had positive association (e.g. proc1p vs. proc2p had r∼0.39). The average correlation coefficients of within-process and between-process in L1 and L2/3 were summarized in With our imaging configuration of one pixel for ∼1 µmthe soma . At astrc signal . Therefo of ROIs . Both in2+) surges in layer 1 (L1) and layer 2/3 (L2/3) of the somatosensory cortex of adult (>P25) rats in vivo in the absence of sensory input. Continuous imaging of the cortical areas for thirty minutes revealed long lasting (>10 s) spontaneous astrocytic Ca2+ surges with a frequency below one per ten minutes. We found there were twice as many “active astrocytes” (astrocytes with at least one Ca2+ surge) in L1 than L2/3. The spontaneous Ca2+ surges under our experimental condition were not influenced by the background neural activity and appeared to be originated intrinsically. Furthermore, process-level Ca2+ dynamics among different branches arising from an astrocyte appeared to be independent in L1, while those in L2/3 had significant synchrony. These results suggest that astrocytes in L1 and L2/3 have distinct modes of activity depending on their surrounding cytoarchitecture. These findings are discussed in detail below.We investigated spontaneously occurring astrocytic calcium  2) was more than ten fold smaller. In fact, no nociceptive effects were reported using adult mice when small craniotomy was made One potential interpretation of the layer differences of astrocytic calcium dynamics is that the preparation of cranial window had nociceptive effects on astrocytes in the superficial L1 which resulted in turning the astrocytes to be ‘reactive’, which is indicated by increased expression of glial fibrillary acidic protein (GFAP) reactive . We havereactive . These r2+ activity 2+ surges than L2/3  could be achieved because an astrocyte has been shown to have processes more than 60 µm occasionally 2+ surges are evoked by ligands that are secreted by a more diffuse secretory system like circulation and astrocytes that express sufficient amount of corresponding receptors.We found that temporal synchrony of spontaneous Cally weak  and therlly weak . These rsionally . In factincrease . Howeverincrease . Considetrocytes  and 8, ttrocytes . Possibl2+ dynamics in processes of an astrocyte in L1 were independent, while that in L2/3 showed higher degree of synchrony  were used for this study. Animals were deeply anesthetized with 1.7 g/kg urethane and the body temperature was maintained at 37.5 degrees with a regulated heating pad  throughout the surgery and following imaging experiment. Physiological saline containing dextrose (5% w/v) was injected subcutaneously as necessary (up to 10 ml/kg/hr) to maintain fluid balance. After the skull was exposed, a metal frame was attached to the skull using a dental acrylic . A craniotomy (diameter 1.0∼1.5 mm) for imaging, centered at the stereotaxic coordinate of anterior-posterior ∼−2.5 mm and medial-lateral ∼3.0 mm from the bregma, was made using a dental drill above the somatosensory cortex. The dura mater was surgically removed. Care was taken to avoid any damage to the pial vessel and cortical surface. Another small craniotomy (diameter 0.5 mm) was made approximately 2.0 mm posterior to the imaging window for intracranial electroencephalogram (EEG) so that the electrode will not make a physical intrusion to the imaged area, while measuring the population activity in the vicinity of the imaged area. The craniotomies were perfused with warmed Hepes-Ringer solution (∼35°C) to maintain surface temperature of the brain during the experiment. All experimental protocols were approved by the RIKEN Institutional Animal Care and Use Committee.2+ sensitive fluorescence indicator Oregon Green 488 BAPTA-1 (OGB-1) and astrocyte specific fluorescence marker Sulforhodamine 101 (SR101) Multi-cell bolus loading was performed to load neurons and glial cells with CaA quartz glass pipette (resistance 2∼3 MΩ) containing physiological saline was inserted 300∼400 µm below the pial surface with an insertion angle of 30 degree. EEG was measured by taking a differential signal between the microelectrode and a reference signal at the neck of the animal. The EEG signal was amplified by 1,000 fold and bandpass filtered between 0.1 and 4k Hz . Electrocardiogram (EKG) was monitored continuously using metal electrodes by taking a differential signal between chest electrode and reference electrode located at a foot of the subject. EEG, EKG, and a synchronizing signal from the two-photon microscope were simultaneously digitized at 25 kHz using a commercial data acquisition card . The data acquisition software was written with LabView .After the imaging measurements were made, the subjects were transcardially perfused with physiological saline, followed by 100 ml of 0.1 M phosphate buffer containing 4% paraformaldehyde. Coronal sections corresponding to the imaged area were cut at 60 µm thickness in 0.1 M phosphate buffer (pH 7.4) using a vibratome . For the detection of cytoarchitectural difference of cortical layers , slices http://rsb.info.nih.gov/ij/). The values were exported to MATLAB  and the fluorescence change was computed. Ca2+ surges of astrocyte were detected when fluorescence intensity of OGB-1 reached higher than 117% of baseline fluorescence intensity. Baseline fluorescence intensity was defined as the mean of the lower two-third of the fluorescence intensity histogram for each astrocyte during the thirty minute imaging. Duration of each Ca2+ surge was defined as duration of fluorescence intensity above 50% of its peak intensity during a Ca2+ surge. Pairwise crosscorrelograms between pairs of astrocytes were computed by standardizing the fluorescence signal to be the Z score, so that the height of the crosscorrelogram represents the Pearson correlation coefficient. Synchrony of Ca2+ traces of a pair of astrocytes was calculated using the Fisher's exact test in which traces of fluorescence intensity of Ca2+ indicator was divided into two periods, i.e. periods with or without Ca2+ surge, for each imaging frame. For astrocytic process analyses, the Pearson correlation coefficients were computed to assess the synchrony of Ca2+ dynamics. All data are expressed as mean±standard deviation (SD), except where otherwise noted. The Student's t-test, Wilcoxon signed-rank test (for two groups) and ANOVA, followed by the Tukey multiple comparison test (for three or more groups) were used to determine the statistical significance (p<0.05) of differences. Statistical analyses were performed by programs written with MATLAB.Fluorescence signal was quantified by measuring the mean pixel value of a manually selected somatic area for each frame of the image stack using ImageJ software  in (A). Small red dots indicate period of Ca2+ surge. Vertical position of each trace was adjusted arbitrary to improve visibility. Scale bar: A, 100 µm; B, 50%.Representative Time Course of Spontaneous Ca(1.90 MB TIF)Click here for additional data file.Figure S22+ surge was suppressed by PPADS. (B1) Average traces±SEM (n = 8) of astrocytic Ca2+ responses to ATP in the presence (red) or absence (black) of PPADS. ATP (100 mM) was pressure-injected into the somatosensory cortex using a pipette . (B2) Comparison of peak amplitude of ATP-induced Ca2+ surges of astrocytes. PPADS significantly reduced the Ca2+ responses . C, Atropine prevented EEG desynchrony induced upon a stimulation of the nucleus basalis (NB). (C1) Representative EEG traces of somatosensory cortex with NB stimulation in the absence (upper trace) or presence (lower trace) of atropine. Immediate after NB stimulation, slow wave transiently disappears for a time period of three to five seconds (single arrow), while slow wave persists after NB stimulation in the presence of atropine (asterisk). NB was stimulated with a bipolar tungsten electrode . (C2) The effectiveness of NB stimulation was quantified by measuring ratio of EEG amplitude 1∼3 sec prior to and after NB stimulation. Values larger than 1 indicate decrease in EEG amplitude by NB stimulation. Atropine significantly reduced effectiveness of NB stimulation .A, Amplitude of EEG was increased by MPEP. (A1) Representative EEG traces of somatosensory cortex before and after MPEP injection. Rats were anesthetized with urethane of dosage:1.2 g/kg) to induce desynchronized state of EEG . (A2) Comparison of power spectrum densities before (black) and after (red) MPEP injection. Traces represent mean±SEM (n = 5). MPEP significantly increased the power of slow wave (0.5–2 Hz) . B, ATP-induced Ca(1.02 MB TIF)Click here for additional data file.Figure S32+ Activities of Processes of an Astrocyte in Layer 2/3. A, Soma and processes of an astrocyte in layer 2/3 were identified with astrocyte specific SR101 image. ROIs were selected in the same manner as 2+ indicator (OGB-1) signal is plotted for each ROI within a single astrocyte in L2/3. ROIs were selected in the same manner as Representative Traces of Ca(1.77 MB TIF)Click here for additional data file.Video S1Imaging of Spontaneous OGB-1 Fluorescence Dynamics in Cortical Layer 1 In Vivo. The movie was taken in the layer 1 (47 µm below the pia) of the primary somatosensory cortex loaded with the Ca2+ sensitive fluorescence dye OGB-1 (green) and the astrocyte specific marker SR101 (red). The movie represents the same area as (8.73 MB MOV)Click here for additional data file.Video S2Imaging of Spontaneous OGB-1 Fluorescence Dynamics in Cortical Layer 2/3 In Vivo. The movie was taken in the layer 2/3 (192 µm below the pia) of the primary somatosensory cortex stained with OGB-1 (green) and SR101 (red) for thirty minutes. The movie represents the same area as (9.97 MB MOV)Click here for additional data file.


However, the mechanism responsible was unclear.Diminished calcium (Ca3R) and the SR Ca2+ pumps (SERCA 2 and 3). Generally, a decrease in IP3R levels and dramatic increase in ryanodine receptor (RyR) levels were noted in the aortic samples from diabetic animals. Redistribution of the specific IP3R subtypes was dependent on the rat model. SERCA 2 was redistributed to a peri-nuclear pattern that was more prominent in the DR-BB diabetic rat aorta than the STZ diabetic rat. The free intracellular Ca2+ in freshly dispersed VSMCs from control and diabetic animals was monitored using ratiometric Ca2+ sensitive fluorophores viewed by confocal microscopy. In control VSMCs, basal fluorescence levels were significantly higher in the nucleus relative to the cytoplasm, while in diabetic VSMCs they were essentially the same. Vasopressin induced a predictable increase in free intracellular Ca2+ in the VSMCs from control rats with a prolonged and significantly blunted response in the diabetic VSMCs. A slow rise in free intracellular Ca2+ in response to thapsigargin, a specific blocker of SERCA was seen in the control VSMCs but was significantly delayed and prolonged in cells from diabetic rats. To determine whether the changes were due to the direct effects of hyperglycemica, experiments were repeated using cultured rat aortic smooth muscle cells (A7r5) grown in hyperglycemic and control conditions. In general, they demonstrated the same changes in protein levels and distribution as well as the blunted Ca2+ responses to vasopressin and thapsigargin as noted in the cells from diabetic animals.VSMCs from autoimmune type 1 Diabetes Resistant Bio-Breeding (DR-BB) rats and streptozotocin-induced rats were examined for levels and distribution of inositol trisphosphate receptors (IP2+ signaling in VSMCs from diabetic animals is related to decreases and/or redistribution in the IP3R Ca2+ channels and SERCA proteins. These changes can be duplicated in culture with high glucose levels.This work demonstrates that the previously-reported reduced Ca While improved care for people with diabetes has reduced the overall risk of complications and mortality, rates of mortality for people with diabetes is still two fold higher than those without diabetes ,2. Cardi2+) from intracellular stores, including the sarcoplasmic reticulum (SR).While the majority of research effort concerning diabetic vascular disease has been focused on changes in the endothelial cells, alterations in the vascular smooth muscle cells (VSMCs) are also known to occur -13. The 2+ stores; the inositol 1,4,5-trisphosphate (IP3)-sensitive store and the ryanodine-sensitive store [3 release of Ca2+ from intracellular stores is a common pathway for activation of many proteins including PKC [2+ with diabetes are inconsistent, but Ca2+ transients in response to agonists such as angiotensin II are clearly attenuated in VSMCs from diabetic animals [3-induced Ca2+ responses could be due to changes in the intracellular Ca2+ channels, the intracellular Ca2+ storage site (SR), or the sarcoplasmic/endoplasmic Ca2+ ATPase (SERCA). This study examines changes in the intracellular Ca2+ regulatory proteins in two animal models of type 1 diabetes. The streptozotocin-induced diabetic rat is a well-established rat model using a toxin to destroy the pancreatic beta cells. The second model utilizes the DR-BB rat, which better mimics the autoimmune component of type 1 diabetes. Results from these animal models were compared to a cell culture model (A7r5) with hyperglycemia.In VSMCs there are two channels that have been identified as important in the release of intracellular Cave store . In VSMCding PKC  and PLC. animals . The bluSeven week old male Sprague Dawley rats  weighing 250-270 grams were injected intraperitoneally with 65 mg streptozotocin  per kg body weight (n = 15). Non-diabetic control rats (n = 12) were injected with vehicle (citrate buffer). Blood glucose levels greater than 250 mg/dL, measured by an Accu-Check Advantage glucometer , indicated the development of diabetes.The blood glucose levels and body weights of all rats were assessed weekly. The final weight and blood glucose levels are shown in Table Diabetes was induced in 21-26 day old Diabetes Resistant Bio-Breeding (DR-BB) Wistar rats using anti-RT6 monoclonal antibody  DS4.23 hybridoma in 2 ml of tissue culture medium injected 5 times/week in combination with polyinosinic-polycytidylic acid (Sigma), a non-specific immune system activator at 5 μl/g of body weight 3 times/week. Induction treatment was discontinued at the onset of diabetes as determined by a blood glucose level above 250 mg/dL. Diabetes typically occurred 2-3 weeks after the treatment was initiated. Within 1 week of diabetes development, the diabetic rats were treated with insulin  through subcutaneous implantation of an insulin-filled Alzet osmotic pump . Blood glucose was closely monitored for the remaining time, and hyperglycemic animals were given insulin injections as needed in amounts based upon the severity of hyperglycemia. Control rats underwent the same manipulations except pumps were filled with saline. Body weight and blood glucose were followed weekly, with the final values shown in Table Dissected aortas and femoral arteries were cleaned of adventitia in cold phosphate buffered saline (PBS) buffer. The vessels were cut lengthwise to expose the intima, which was gently swabbed to remove endothelial cells. Each vessel was pinned, lumen side up, in a small covered beaker containing Sylard Gel  and bathed in collagenase solution containing deoxyribonuclease I, collagenase type 2, soybean trypsin inhibitor  and bovine albumin (Sigma). After 30 minutes of shaking at 37°C, the first digest was discarded as it contained predominantly endothelial cells. New collagenase solution was added and further dispersions were gathered every 2 hours. Cells were gently centrifuged to remove the enzyme solution before being rinsed and re-suspended in media.2. Cells were grown in flasks, then seeded in 100 mm dishes or on coverslips with glucose concentrations of either the supplier-recommended concentration  or high glucose (75 mM). Because 25 mM glucose is high compared to physiological serum levels, a third (low) glucose concentration was tested (5.5 mM). The effects of osmolarity were controlled by the addition of mannitol. Cells were grown to semi-confluence prior to testing.Cells from the A7r5 cell line , derived from rat thoracic aorta, were grown in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C, 5% CO3Rs). After electrophoretic separation, proteins were transferred from a gel to a PVDF membrane (Pierce) overnight. Ponceau's stain (Sigma) was used to assess protein loading prior to blotting. A standard immunoblotting protocol was performed. Primary antibodies to SERCA2, SERCA3, IP3R type I and RyR , and IP3R type 2 and IP3R type 3 (Santa Cruz) were used. GAPDH was used as a housekeeping gene product . Anti-HSP90 (Santa Cruz) was used to assess the purity of the cytoplasmic extractions and anti-p62  or anti-USF-2 (Santa Cruz) were used to test the purity of the nuclear extracts. Detection was performed using enhanced chemiluminescent reagent (Pierce) or SuperSignal West Pico Chemiluminescent reagent (Pierce). Duplicates were made of each immunoblot experiment.Total proteins were extracted from tissue or cell culture using lysis buffer containing 10 mM Tris, pH 7.4, 1 mM sodium ortho-vanadate and 1% SDS. To separate nuclear and cytoplasmic proteins, the NE-PER kit  was used. Each sample was concentrated by ultrafiltration using Microcon YM-10 . Protein concentrations were measured in 96-well format using the BCA kit  and a MRX microplate reader . A total of 30 or 50 μg of protein was loaded in each lane of 8, 10 or 4-15% gels. Rat cardiac tissue extract was used as a positive control for ryanodine receptor (RyR) and A-10 cell lysate  was used for Inositol 1,4,5 trisphosphate receptors . Fixed cells were rinsed with PBS, permeabilized with 1% Triton X-100 (Sigma) and rinsed well before blocking in 10% goat or donkey serum . Cells were incubated with primary antibody (same as above) diluted in 5% non-fat dry milk  solution overnight in a cold box at manufacturer recommended concentrations. Alpha smooth muscle actin (Sigma) was used to identify SMC type. Cells were repeatedly rinsed in PBS before incubation with secondary antibody conjugated to Rhodamine Red or Cy 2 (Jackson ImmunoResearch) in a dark chamber. Brefeldin A  was used to evaluate sarcoplasmic reticulum (SR). Coverslips were rinsed and allowed to air dry in the dark before mounting. Images were captured using an Olympus Fluoview 300 Confocal Microscope with the consistent confocal settings for each pair of control and diabetic cells per single primary antibody. Experiments were performed in triplicate.2+ sensitive fluorophores, Fluo-4/AM and Fura-Red/AM (invitrogen) by incubation at 37°C for 30-60 minutes. The coverslips were inserted into an Attofluor chamber (Molecular Probes) and rinsed with warm PBS. PBS with ethylene glycol tetraacetic acid (EGTA) to buffer extracellular Ca2+ (0 Ca2+), was added to the chamber mounted on the stage of a confocal microscope so that the measured Ca2+ changes were limited to the intracellular stores only. Baseline measurements were taken on quiescent cells for 3-4 minutes prior to the application of any agonist. Vasopressin  or thapsigargin  were added to the bath at the times indicated in the results section. Images were collected every 5 or 10 seconds using an inverted Nikon Eclipse TE 300/Bio-Rad MicroRadiance Plus Laser Scanning Confocal Microscope  or an Olympus Fluoview 300 confocal microscope . Fluoview software was used to analyze Ca2+ signaling experiments. Time-to-peak was defined as the time of agonist application to the time the maximal magnitude of the response was obtained. The duration of the Ca2+ signal was defined as the time from the upstroke of the response to the return within 25% of initial baseline value.Freshly dispersed VSMCs or cultured A7r5 cells were allowed to attach to coverslips for 1 hour in PBS at 37°C before loading with 1 μM each of two Ca2+ signaling experiments. Adobe Photoshop  was used to analyze relative fluorescence intensity, and color analysis. Analysis of variance was performed for Ca2+ fluorescence and immunoblot density values using Sigma Stat software. When appropriate, statistical differences were assessed using Dunnett's test for multiple comparisons after a one-way analysis of variance using Sigma Stat . A probability level of p < 0.05 was defined as a significant difference.All data points were normalized for background signal prior to statistical analysis. Fluoview software  was used to analyze CaThe blood glucose levels were significantly higher p < 0.001) in both diabetic rat models. However, the animals from the toxin-induced diabetes (STZ) were significantly more hyperglycemic when compared to the genetically-prone diabetic group (DR-BB) . With diabetes, the resting Ca2+ concentration declined in both the nucleus and the cytoplasm; however, the nuclear decline was greater so that quiescent cells from diabetic animals had similar nuclear and cytoplasmic Ca2+ concentrations . The amplitude of the nuclear thapsigargin response was not different in VSMC from control or diabetic animals using either animal model . These findings are opposite of those measured in cells from diabetic animals (either model) and may explain some of the confusion in the field regarding changes in resting cytoplasmic Ca2+ concentrations with diabetes. There was a significant difference between the high glucose condition and the other two conditions (p < 0.0001). There was no statistically significant difference in the free intracellular Ca2+ between the low glucose condition and the medium glucose condition. In fact, there was no statistical difference between the low and medium glucose conditions for all assays tested. Therefore, only medium and high glucose conditions are reported in this study.To determine whether the blunted agonist responses were due directly to the exposure to hyperglycemia or were a physiological adaptation to long term diabetes, cultured rat aortic VSMCs were grown in high glucose. Figure 2+ that was diminished by high glucose conditions. Figure 2+ levels made it difficult to determine a change in vasopressin- induced peak responses. In order to compare the amplitude of the responses, the differences in baseline Ca2+ concentrations were normalized by dividing values by the resting Ca2+ fluorescence values (F/Fo) following the methods reported previously [When stimulated with vasopressin or thapsigargin, cells responded with an immediate increase in intracellular Caeviously . The nor2+ signal from within the nuclear compartment. Using ratiometric Ca2+-sensitive fluorophores, the amplitude of the Ca2+ signal to vasopressin stimulation (at time 0) in the nuclear compartment was shown to be decreased when A7r5 cells were grown in high glucose  displayed 99% synchrony in the nuclear and cytoplasmic Ca2+ signals produced by vasopressin or thapsigargin. Asynchrony between the nuclear and cytoplasmic Ca2+ transients with stimulation was significantly increased in cells grown in high glucose, resulting in 13% of the total cells demonstrating asynchronous behavior (n = 138 cells).Under medium and low glucose conditions, the timing of the Ca2+ transients well above 100% of baseline fluorescence levels in basal conditions  or low (n = 34 cells) glucose.In addition to asynchronous behavior in response to an agonist, 17% of the A7r5 cells exposed to high glucose demonstrated large magnitude spontaneous nuclear Ca2+ signals caused by diabetes and hyperglycemia, intracellular Ca2+ regulatory proteins including Ca2+ pumps and channels were measured for protein levels and localization. Resting intracellular Ca2+ levels in VSMCs are maintained by plasma membrane and intracellular Ca2+ pumps (SERCA). The amount of the SR Ca2+ pump (SERCA2 and SERCA3) protein in aortic VSMCs was significantly lower with diabetes in both animal models tested. Figure In order to explain the dramatic changes in Ca2+ channels and pumps when A7r5 cells were grown in medium or high glucose conditions. In immunoblotting experiments, the amount of SERCA2 protein in the cytoplasmic fraction remained unchanged with high glucose, but SERCA2 levels increased in the nuclear extract with high glucose treatment, although it is important to note that the nuclear SERCA2 levels were still low with high glucose exposure. In low and medium glucose conditions, there was no detectible SERCA2 in the nuclear fraction, but in high glucose a clear band appeared. In contrast, SERCA3 was detected primarily in the nuclear extracts with cytoplasmic bands faintly visible. There was an average of a 60% decrease in the amount of SERCA3 in the nucleus with high glucose treatment. The majority of HSP-90 was found in the cytoplasm while USF-2 was strictly nuclear, validating the purity of the nuclear and cytoplasmic extracts (not shown).Figure 2+ regulating proteins, immunofluorescence provided more detailed information. SERCA2 immunostaining illustrated the intricate web of SR with a more pronounced perinuclear staining in cells from the DR-BB rats  and the ryanodine receptor (RyR). There are three subtypes of IP3Rs, and each was examined in the aorta of the diabetic animal models and in cultured cells exposed to hyperglycemic conditions. Type 1 IP3Rs (IP3R-1) showed a significant decline in levels in the aortas from both models of diabetes with a 69% decline in the DR-BB diabetic animals and a 53% decline in the STZ rats  were only tested in the genetically-prone DR-BB model of diabetes, showing a significant decline with diabetes. IP3R type 3 (IP3R-3) levels were not detected in amounts allowing immunoblot analysis of protein levels in either animal model.The channels responsible for releasing Cas Figure , Table 33R prompted investigation of the other major SR Ca2+ release channel in smooth muscle cells, the RyR to determine whether its subcellular distribution changed, possibly in response to changes in the IP3R. We hypothesized that the RyR could be either down-regulated due to a decline in cellular Ca2+ signaling or upregulated as a compensatory mechanism for the decline in IP3R protein levels and cellular signaling. The RyR, increased dramatically with diabetes, regardless of the animal model  in cellular fluorescence intensity in the VSMCs from diabetic aortas (not shown) and from cultured A7r5 cells  and when cultured in hyperglycemia . This was true of both diabetic animal models and A7r5 cells grown in high glucose concentrations. Immunostaining of the RyR demonstrated primarily patchy cytoplasmic staining with strong perinuclear localization Figure . A predo3R subtypes, RyR, and SERCA were similar to other studies using primary aortic SMCs [3R-1, SERCA2 and SERCA3 expression in a manner very consistent with our results from VSMCs.VSMC dysfunction and subsequent disease is the main cause of mortality in type 1 diabetes, but the underlying mechanisms are largely unknown. In normal (medium) glucose conditions reported here, the distribution of the IPtic SMCs , rabbit tic SMCs , and cultic SMCs . Studiestic SMCs , islet ctic SMCs  using di2+ storage and release in aortic and femoral VSMCs. These changes lead to altered Ca2+ signaling in the cytoplasmic and nuclear compartments in response vasopressin. VSMCs from two different diabetic rat models showed a decline in the levels of IP3R and SERCA protein with diabetes. Changes in the distribution of the IP3R subtypes and SERCA subtypes were dependent on the rat model, but fully supported the immunoblot results of overall decreased amounts. To measure the relative role of hyperglycemia on the above mentioned changes, rat aortic cultured cells (A7r5) were exposed to various concentrations of glucose. Like the freshly dispersed cells from the diabetic animals, cultured cells exposed to high glucose had blunted Ca2+ responses to vasopressin and decreased levels of Ca2+ regulatory proteins. Thus, the changes noted in vSMCs from diabetic animals were likely due to exposure of the vSMCs to hyperglycemic conditions rather than adaptation of the cells to the complex disease of diabetes. This is the first study to report of both diminished levels and subcellular redistribution of key Ca2+ regulatory proteins in cultured and freshly dispersed VSMCs.The results of this study clearly demonstrate changes in the regulatory proteins associated with intracellular Ca2+ response in VSMCs from diabetic animals [2+ oscillations. Such oscillations in A7r5 cells have been reported to be associated with intracellular Ca2+ stores and entry of Ca2+ from the extracellular milieu [2+ sparks occurring around the nucleus of primary VSMCs [2+ release events as all experiments were performed in zero extracellular Ca2+.Previous studies have described a blunted Ca animals -30. Uniqr milieu ,22. Othery VSMCs . The effIn order to thoroughly examine the effects of hyperglycemia on VSMC function, two different animal models of type 1 diabetes were utilized; the toxin-induced STZ diabetic rat and the immune/inflammation model of type 1 diabetes, the DR-BB rat. The differences noted between the two models were minor, but in general the diabetes induced changes were more robust in the DR-BB rats. It is possible that any small differences we noted between rat models were a function of the rat strain, the method of diabetic induction, or the administration of insulin to the DR-BB animals.2+ from the cytoplasm is dependent on the SERCA proteins on the SR and nuclear envelope, estimated to be responsible for over 40% of the Ca2+ buffering in VSMCs [2+, SERCA inhibits migration of VSMCs into the intima of the vessel [2+-ATPase of the SR in VSMCs and is thought to be most important in cytoplasmic Ca2+ buffering [2+ transient suggest that the decrease in protein levels affected the ability of the cells to buffer Ca2+ via SERCA as has been shown in other labs [In VSMCs, much of the ability to remove Cain VSMCs ,34. Impoe vessel , a commoe vessel , we did uffering . Changesher labs .2+ from the cytoplasm following stimulation [The function of SERCA3 in VSMCs still remains elusive. Originally SERCA3 was reported to be absent from muscle cells. However, thorough examination of tissues with subtype-specific antibodies, revealed that at least one member of the SERCA3 family was equally expressed in all tissues tested . In humamulation . Given smulation .2+ responses were tested in VSMCs from both the aorta and the femoral arteries. The diabetes-induced changes in protein levels and in the Ca2+ responses were the same in VSMCs from either location with the exception of SERCA2 levels, which were increased in the femoral artery, but decreased in the aortic VSMCs in the STZ rat. This increase in the femoral artery agrees with a study finding increased SERCA activity and levels in resistance arteries from diabetic dyslipidemic pigs [Vascular reactivity to vasopressin in diabetes varies between vascular beds ,43. For mic pigs . SERCA2 2+ channel activity, the results presented here build on earlier work by Ma et al., illustrating a decline in IP3 responses in VSMCs from STZ treated rats [2+ responses via IP3 receptors and Ca2+ influx across the plasma membrane. A decrease in IP3R-1 levels in diabetic arteriolar SMCs has been published previously [2+ regulatory proteins on the SR by conducting live cell experiments in zero extracellular Ca2+ conditions. Further, we showed that the findings were not dependent on the mode used to induce diabetes, using either a toxin-induced rat model or genetically-bred animals, as the results were generally consistent in the aorta and femoral arteries of the STZ and DR-BB animals. The results suggest that the decreased activity in the VSMCs associated with diabetes is due to shared changes in the SR Ca2+ release channels (IP3R) and SERCA resulting in diminished Ca2+ transients, especially in the nuclear compartment. In addition to selectively activating genes, nuclear Ca2+ may also play an important role in stabilizing mRNA in a variety of cell types [2+ responses in VSMCs as a result of diabetes is a question that should be intensely pursued, given the findings of this study.When analyzing the Cated rats . The Ma eviously . The curll types . The fun2+ regulation. Previous studies have shown direct effects of glucose on cultured cells, but have typically worked with primary cultures [2+ in the extracellular media was sufficient to elicit a response similar to that seen in cells from diabetic animals. Due to the fact that we utilized a cultured cell line in which the normal growth media contained 25 mM glucose (referred to as medium in this study), we had to increase the glucose concentration dramatically to evoke a diabetes-like response. While the sensitivity to glucose was different in the culture cells, the other characteristics of the glucose-induced change in freshly dispersed and cultured cells were exceedingly similar.The similarity of the results from identical experiments conducted in freshly dispersed cells from diabetic and control rats, and cultured rat aortic cells grown in normal and high glucose are striking and underlie the importance of chronic exposure to glucose as a major inducer of changes in intracellular Cacultures ,48,35. I2+ response from cytoplasmic may not be immediately clear. We are not the first to report on selective Ca2+ signals in the nuclear that were unique from the cytoplasm as Bkaily et al demonstrated in neurons [2+ have been reported to be regulated independently in several cell types [2+ signaling around and within the nucleus of smooth muscle cells have led some to suggest that clusters of lysosomes with high densities of RyR form in the perinuclear region forming physical junctions with the SR to comprise a trigger zone of Ca2+ [2+ signal with high glucose is unknown, but it is likely that VSMCs have nuclear-specific Ca2+ transcription regulated pathways just as neurons have been shown to possess [The importance of separating the nuclear Ca neurons ,50. Nuclll types ,52. Zone of Ca2+ . The res possess ,55.3-induced Ca2+ transients noted previously in VSMCs from diabetic animals. This is likely due to the altered distribution and/or levels of Ca2+ regulatory proteins including the IP3R, RyR, and SERCA. Determining how diabetes changes protein distribution and amounts is paramount, as these changes clearly have downstream effects within the cells that may be linked to cell proliferation and migration.While diabetes is a complex disease that includes hyperglycemia, hypoinsulinemia or hyperinsulinemia and numerous adaptations in metabolic proteins as well as changes in regulatory factors, it appears that high glucose is predominantly responsible for the decreased IPThe authors declare that they have no competing interests.YS carried out the confocal experiments and protein analysis as partial completion of her dissertation requirements. IS oversaw all immunoblot experiments and their analysis. RL was responsible for all animal work and participated in the design of the experiments using the DR-BB and STZ rats. LSB oversaw all work and wrote the manuscript. All authors read and approved the final version of the manuscript.