Analgesia is often indicated in rabbits undergoing surgical procedures or suffering from various painful conditions and the most common adverse effects associated with NSAIDs occur in the gastrointestinal tract (GIT). The objective of this study was to determine the potential effect of long-term (21 days) meloxicam administration on the fecal bacterial microbiota in healthy rabbits.Samples of hard feces were collected from six rabbits treated with meloxicam  on days 0,6,14 and 21. Next generation sequencing of V4 16S rRNA gene products was performed.P = 0.02).A total of 2589912 V4 rRNA gene sequences passed all quality control filters. Firmicutes predominated (82.0 ± 6.2%). Sixteen other phyla were also identified but other than Verrucomicrobia (4.4 ± 4.9%), all accounted for less than 1% of the identified sequences. Within Firmicutes, Clostridia was the dominant class, accounting for 76% of operational taxon units (OTUs). In general, there were only few differences observed between time points and different rabbits at the phylum level. A significant change was observed in the relative abundance of Proteobacteria over the 4 time points (The gastrointestinal tract of rabbits harbors dense and diverse microbiota. Significant alteration of the hard fecal microbiota does not appear to be a considerable adverse effect expected in rabbits treated for 21 days with oral meloxicam at a dose of 1 mg/kg. Analgesia is a basic aspect of veterinary medicine, indicated in both laboratory and pet-owned animals undergoing surgical procedures or suffering from various painful conditions . One of The fecal or intestinal microbiota of domestic rabbits has been extensively studied through culture dependent methods, as well as earlier generation culture-independent methods, with a focus on the cecal microbiota -13. HoweThis study explores the stability over time of bacterial communities in hard feces in healthy rabbits medicated with oral meloxicam, using high throughput sequencing of the V4 region of the 16S rRNA gene.Pasteurella sp.) free. The rabbits were housed individually at the research facilities of the Kansas State University College of Veterinary Medicine at a constant temperature (21°C) and humidity (60%), and were exposed to cycles of 16 hours of light and 8 hours of dark per day. Rabbits were fed free choice of timothy haya, timothy-based pelleted dietb and water was available ad libitum. Rabbits were acclimated to the facility for 5 days after their arrival and were habituated to handling prior to initiation of the study. Immediately prior to the start of the study, each rabbit was determined to be behaviorally normal and healthy based on a thorough physical examination and clinical pathology testing , with results compared to published reference ranges [c (100 mg/kg Intravenous), and necropsy was performed within 15 minutes of euthanasia.Ethical approval was obtained from the Institutional Animal Care and Use Committee of Kansas State University. Rabbits enrolled in this study were part of a broader study evaluating long term (29 days) administration of oral meloxicam. Six clinically normal 3-month-old intact female New Zealand White rabbits  were included in this study. The rabbits were obtained from a commercial source and were specific pathogen (e ranges . Physicad (1 mg/kg) was orally administered daily for 29 days to each rabbit via a 3-mL syringe. The rabbits’ behaviour, attitude, mentation, level of activity, eating and drinking, and defecation were subjectively assessed daily by the researchers. Hard fecal samples were collected prior to treatment (day 0) and on days 6, 14 and 21 from the start of the meloxicam treatment. At each time point morning (08:00), 10 fecal pellets from a fresh elimination were placed into an individual collection bag. Therefore, four fecal samples were collected from each rabbit during the study period for determination of fecal microbiota. Each sample  was weighed using a digital gram scale and each fecal pellet was measured using hand calipers. All bagged samples were stored at −70°C until analysis.Meloxicame following the manufacturer’s “stool DNA protocol for pathogen detection”, which includes an initial bead beating step and proteinase K treatment. DNA quantity and quality were accessed by spectrophotometry using the NanoDropf.DNA extraction was performed using a commercial kitg, 1.3 μL of BSAh, 0.5 μL of each 16S primer (1000 pmol/μL) and 1 μL of each Illumina primer (1000 pmol/μL). The mixture was subjected to the following PCR conditions, 3 minutes at 94˚C for denaturing, and 35 cycles of 45 seconds at 94˚C for denaturing, 60 seconds at 50˚C for annealing and 90 seconds at 72˚C for elongation followed by a final period of 10 minutes at 72˚C and kept at 4˚C until purification.PCR amplification of the V4 region of the 16S rRNA gene was designed based on Klindworth et al.  using thi by mixing 22 μL of amplicon with 72 μL of AMPure on a 96 well plate. After 5 minutes at room temperature, beads were separated and washed twice with 80% ethanol and eluted in 30 μL of water. After purification samples were quantified by spectrophotometry using the NanoDropj and normalized to a final concentration of 2 nM. Sequencing of the library pool was performed at the University of Guelph’s Advanced Analysis Centre using an Illumina MiSeqk and 2x250 chemistry.PCR products were evaluated by electrophoresis in 2% agarose gel and purified with the Agencourt AMPure XPThe MOTHUR package of algorithms (v1.30) was used for analysis . Paired Sequences were binned into OTUs at a 3% (0.03) dissimilarity level, and taxonomy was assigned using the Silva 16S rRNA reference database .Coverage was assessed using Good’s coverage. Population diversity was described using the inverse Simpson’s index. Relative abundances were compared between animals and between timepoints using ANOVA.Dissimilarity between the two groups was assessed through creation of dendrograms using the Yue & Clayton measure of dissimilarity  and traditional Jaccard index . Parsimony and unweighted unifrac  tests weMeloxicam was easily administered daily to each rabbit, and all of the rabbits seemed to remain healthy during the study. Subjectively, none of the rabbits had changes in behavior, attitude, mentation, level of activity, amount of food or water consumed, or fecal production. Objectively, all participating rabbits continued to gain weight  when compared to the beginning of the study period. Also, all values in the weekly serum biochemistry analysis were within normal limits both before and during the study. No changes were observed in hard feces’ shape, color and consistency (no diarrhea or ileus). The mean weight of a 10-pellet fecal sample was 6.75 g (range 6–9 g) and mean measured individual fecal pellet size was 6.2 mm (range 5–8 mm). Post-study gross necropsy and histopathologic evaluation of all participating rabbits did not reveal any major abnormalities.A total of 3453182 V4 16S RNA gene sequences were obtained; 2589912 of which passed all quality control filters. The number of sequences per sample ranged from 35178 to 250720 . Further analysis was performed on random subsampling of 35178 sequences per sample. Excellent sample coverage was obtained with this subsampled population, as demonstrated by a mean Good’s coverage value of 0.972  and rarefaction curves  of sequences Figure . SixteenWithin Firmicutes, Clostridia was the dominant class, accounting for 76% of OTUs from Firmicutes, with Ruminococcaceae and Lachnospiraceae being the main families , representing 48 and 20% of sequences from Clostridia, respectively. Unclassified Clostridia were common (24%), followed by smaller numbers of Bacilli (2.9%).Akkermansia and Persicirhabdus were the two identified genera, but they only accounted for 9.9 and 0.2% of sequences from Verrucomicrobia, respectively. The remaining members of that family were unclassified at the genus level.Within Verrucomicrobia, Verrucomicrobiaceae was the predominant (98%) family. Within that family, P = 0.02), with day 21 having a significant higher relative abundance than the other three time points. Otherwise, no significant differences between timepoints were identified.There were few differences between different timepoints and different rabbits at the phylum level. Rabbit 4 had a significantly higher relative abundance of Verrucomicrobia (12%) compared to the others (2.1-3.5%) (P = 0.03). No other differences between rabbits were evident at the phylum level. There was a significant change in the relative abundance of Proteobacteria over the four timepoints , but there was a difference using the Jaccard index (P = 0.011), with significant differences between days 0 and 21 (P = 0.002) and days 14 and 21 (P = 0.030).Principle coordinate analysis results are presented in Figures Rabbits enrolled in this study did not show any abnormal clinical signs, hematological values, postmortem evaluation, or physical changes of the hard feces throughout the observed study period, thus suggesting that the tested meloxicam dose can be considered safe for clinical use.This study identified marked (but perhaps unsurprising) richness and diversity of the fecal microbiome that is incomparable with culture-dependent studies, as an average of over 2000 OTUs were identified. This and the corresponding diversity indices highlight the complexity of the rabbit fecal microbiota, even in rabbits housed under controlled conditions with little or no variation in diet and the need to use broad approaches such as this to understand this environment.The predominance of Firmicutes was not particularly surprising, as this has been reported in other culture-independent studies of the rabbit  and can Akkermansia (a member of this phylum) has been shown to be decreased in obese versus lean children and mice [While the predominance of Firmicutes was not unexpected, the utter dominance of this phylum was remarkable. In studies of the fecal microbiome of most species, while one phylum may be most common, there are typically multiple phyla with relative abundances greater than 5% (or higher) ,29-31. Hand mice ,36, to band mice , to be aand mice . These sThe number of sequences that were unclassified at the phylum level was large, although within the range that has been reported in other fecal microbiome studies and lower than that reported in earlier cloning-based studies of the rabbit cecal microbiota ,39. EvenWhen assessing the microbial composition of these rabbits, there was surprisingly little clustering by individual, as is evident from the dendrograms and PCoA plot. With studies of other species, it is typically reported that there is less intra-individual variation than inter-individual variation ,41, and Little impact of treatment was identified and there were inconsistent results between different tests. Differences were noted between days 0 and 21 using parsimony test and AMOVA, as well as days 14 and 21 using AMOVA. However, these were only when analysis was based on the Jaccard index. It is important to consider differences between these indices. The Yue & Clayton index is a measure of population dissimilarity based on both microbial presence and abundance, while the Jaccard index compares community membership without assessment of the relative abundance of different members of the community. Thus, differences in Jaccard but not Yue & Clayton indices suggest that changes in the community membership (addition or loss of members) occurred, but that this may have mainly involved low abundance members of community based on a lack of difference when relative abundance is considered. Other than the potential long-term microbiota adjustment to a new diet  the accuLimitations of the study must be considered. Since there was no untreated control group raised in parallel with these animals and they were all treated at the same time, it is possible that there was some unidentified factor such as a subtle change in diet or stress that accounted for the microbiota change. Additionally, it is known that the fecal microbiome of laboratory raised rabbits is different from rabbits raised under other conditions  -44. As wThe fecal microbiome of the rabbit is a complex and diverse polymicrobial ecosystem, composed of a wide range of both well-understood and completely unknown bacteria. While there were some noted differences over time, there was no clear and consistent impact of meloxicam treatment on the fecal microbiome. Given the importance of the intestinal microbiome in health and disease, particularly amongst hindgut fermenting species such as the rabbit, it is clear that there is inadequate understanding of the ‘normal’ microbiota, how it varies within and between rabbits, and how this complex polymicrobial ecosystem influences (and is influenced by) disease, nutrition, management and myriad other factors.In the absence of adverse clinical, histologic and significant microbial changes observed in rabbits enrolled in this study, it is suggested that alteration of the hard fecal microbiota is not a significant adverse reaction expected with daily oral meloxicam administrated at the tested dose.http://metagenomics.anl.gov).The data set supporting the results of this article are available at the MG-RAST metagenomics analysis server (project 8102, aWestern Timothy Hay, Oxbow Animal Health, Murdock, Neb.bEssentials – Young Rabbit, Oxbow Animal Health, Murdock, Neb.cVortech Pharmaceuticals, Dearborne, MO.dMetacam 1.5 mg/ml oral suspension, Boehringer Ingelheim Vetmedica, St Joseph, MO.eE.Z.N.A. Stool DNA Kit, Omega Bio-Tek Inc., Doraville, GA.fRoche, Mississauga, ON, Canada.gInvitrogen, San Diego, CA.hInvitrogen, San Diego, CA.iBeckman Coulter Inc, Mississauga, ON, Canada.jRoche, Mississauga, ON, Canada.kIllumina Inc, San Diego, CA.The authors declare that they have no competing interests.DE participated in fecal collection, data generation and interpretation, and manuscript preparation. JSW participated in data analysis and interpretation, and manuscript preparation. Both authors read and approved the final manuscript.


ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts.Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2-octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor.Bradykinin, via B2+]i mobilization through the cooperation of B2 and P2Y12 receptors.Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca Despite its overwhelming size throughout the body, the connective tissue has been generally overlooked or misunderstood. It has been considered as relatively superfluous apart from its supporting role amongst more specialized tissues . It has 2 receptor subtype. In intact cells, bradykinin was shown to induce the activation of phospholipases A2, C and D; the release of prostaglandins, the accumulation of cyclic AMP and of cyclic GMP, and the mobilization of Ca2+ were demonstrated  from the cytosol to the plasma membrane i) rises in human subcutaneous fibroblasts  [Ca2+]i rises were observed at concentrations higher than 1 μM. Bradykinin typically produced a biphasic response , then declined to a plateau of elevated [Ca2+]i which remained fairly constant until drug washout , which is known to deplete intracellular Ca2+ stores following a transient (< 2 min) rise of [Ca2+]i levels i response given that the peptide was used in a concentration (30 μM) near that necessary for saturation of B2 receptors in these cells  fluorescence intensity decay of cells loaded with quinacrine as compared to the control situation in which the cells were challenged with the Tyrode’s solution  increased (p < 0.05) attenuated bradykinin-induced quinacrine destaining  from the incubation medium did not significantly (p > 0.05) affect the fluorescence intensity decay of quinacrine stained ATP granules  significantly  and pannexin-1 (Panx1) are now widely accepted as putative mediators of ATP translocation to the extracellular milieu in non-excitable cells. The expression of Cx43 is characteristic of fibroblasts from multiple tissue origins ,28. Usin2+]i oscillations in human subcutaneous fibroblasts depend on the release of ATP via hemichannels using subtype selective connexin and pannexin-1 inhibitors. Inhibition of Cx36- and Cx50-containing hemichannels with mefloquine  [2+]i rise  attenuated [Ca2+]i response induced by bradykinin (30 μM). Interestingly, the selective Panx1 mimetic inhibitory peptide, 10Panx  [2+]i response , were more effective in depressing the plateau phase of bradykinin (30 μM) response  the fast [Ca2+]i rise induced by the peptide. Confocal microscopy studies demonstrated that 10Panx (100 μM) also attenuated (p < 0.05) bradykinin (30 μM)-induced ATP release from human fibroblasts loaded with quinacrine  ,30 was de Figure Bi. On the Figure Bii, and e Figure Biii, sig, n = 6) , also dee Figure Biv. CBX  Figures Bii-4Biv,2+]i oscillations involved nucleotides-release by exocytosis we used the vesicular transport inhibitor, brefeldin A  differences were found in [Ca2+]i oscillations produced by bradykinin (30 μM) in the absence and in the presence of BFA  and Baf A1 . Confocal microscopy experiments with primary cultures of rat subcutaneous fibroblasts loaded with the calcium sensitive dye, Fluo-4 NW, and then incubated with FM4-64, a membrane-selective fluorescent dye, were used to evaluate vesicle endocytosis and exocytosis in the time lapse mode [2+]i oscillations were not accompanied by measurable changes in FM4-64 fluorescence signals in the same cells  and Panx1 hemichannels, rather than by vesicle exocytosis, may contribute importantly to the plateau phase of bradykinin-induced [Ca2+]i response in fibroblasts cultured from the human subcutaneous connective tissue.In order to test if bradykinin-induced i rise  reduced significantly p < 0.0 the platThe kinetics of the extracellular catabolism of adenine nucleotides  and formation of metabolites in fibroblasts cultured from the human subcutaneous tissue is shown in Figure 2+]i response in human subcutaneous fibroblasts, we tested its effect in the presence of selective P2Y1, P2Y12 and P2Y13 receptors antagonists  significantly (p < 0.05) attenuated the plateau phase of [Ca2+]i rise caused by bradykinin  without much affecting the magnitude of the initial [Ca2+]i rise  were observed in the presence of MRS 2179  and MRS 2211  which selectively antagonize P2Y1 and P2Y13 receptors, respectively -induced [Cas Figure C. Selecty p < 0.0 attenuaty p < 0.0 attenuaty p < 0.0 attenuaty p < 0.0 attenuat2+]i in human subcutaneous fibroblasts treated with adenine nucleotides (1 mM) in the presence and in the absence of extracellular Ca2+ are shown in Figure 2+]i reaching, respectively, 43 ± 4% (n = 8) and 25 ± 8% (n = 3) of the maximal calcium load caused by ionomycin  within 20 sec from drug application. In Ca2+-free medium , ATP- and ADP-induced [Ca2+]i transients were partially but significantly (p < 0.05) attenuated  adenosine (NECA), which non-selectively activates all four adenosine receptor subtypes in the submicromolar range. NECA  had a negligible effect on [Ca2+]i in human subcutaneous fibroblasts when it was applied either alone  difference was found in [Ca2+]i oscillations caused by bradykinin (30 μM) in the presence of NECA   is a recognized anti-nociceptive agent (see e.g.) and plae.g.), yet coe.g.,39). Giv2 receptors, which are constitutively expressed in most of the tissues exhibiting bradykinin sensitivity. Although our experiments were conducted in non-stressful conditions, the involvement of inducible B1 receptors mediating bradykinin effects in human subcutaneous fibroblasts cannot be ruled out i to a peak that depends on Ca2+ recruitment from internal stores, which then declines to a plateau that is sustained by Ca2+ entry from the extracellular compartment. The long lasting elevation of [Ca2+]i seen after the initial [Ca2+]i peak probably results from a receptor- or second messenger-operated Ca2+ current activated by bradykinin, rather than via a capacitative pathway i caused by bradykinin in human subcutaneous fibroblasts. This conclusion is supported by results showing that: (1) human subcutaneous fibroblasts exhibit P2Y12 receptor immunoreactivity, (2) bradykinin induces the release of ATP that is subsequently hydrolyzed into ADP by membrane-bound ectonucleotidases, (3) ADP has a tendency to accumulate transiently in the cultures, given that we found that the extracellular catabolism of ADP into AMP is the rate-limiting step of the ectonucleotidase cascade, and, finally, (4) adenine nucleotides-induced [Ca2+]i rises in human subcutaneous fibroblasts are dependent on extracellular Ca2+.In several cell types ,25, incl pathway . Here, w2+ influx resulting from the cooperation with ADP-sensitive P2Y12 purinoceptors need to be further investigated. Interestingly, bradykinin causes a rapid translocation of all protein kinase C isoforms from the cytosol to the plasma membrane within the time frame (30 s) of ATP appearance in the cultures i response. Because connexin hemichannels are activated by moderate (< 500 nM) [Ca2+]i elevations, these channels may open in response to bradykinin during the initial [Ca2+]i rise [2+]i oscillations as demonstrated by the partial reduction of the initial [Ca2+]i rise of bradykinin in the presence of 2-octanol and CBX.Little is known about the mechanisms upstream the nucleotide release from human fibroblasts despite the importance of connective tissue ATP signaling in the pathogenesis of acute and chronic inflammatory pain ,19. Mult+]i rise  and cont10Panx, which also depressed the plateau phase of the bradykinin-induced [Ca2+]i response in parallel to the decline of ATP release from fibroblasts loaded with quinacrine. Like the effects obtained with apyrase and with the selective P2Y12 antagonist, AR-C 66096, inhibition of hemichannels containing Cx43 and Panx1 were more effective in depressing the plateau phase rather than the peak of the bradykinin response. Taken together, data suggest that Cx43 and Panx1 containing hemichannels have a predominant role on ATP release from human subcutaneous fibroblasts stimulated with bradykinin, thereby instigating the regenerative propagation of intracellular Ca2+ signals. Our findings agree with the observation that mechanically stimulated cardiac fibroblasts release ATP in a CBX-sensitive manner, an effect that the authors attributed to Cx43 hemichannels not excluding a possible involvement of Panx1 containing hemichannels i mobilization from internal stores i-dependent ATP release from human subcutaneous fibroblasts, it appears that VSOR currents would exhibit a slow activation kinetics requiring 15-20 min after bradykinin application to reach a sustained plateau [2+]i signals. Moreover, modulation of VSOR channel permeability through the activation of protein kinase C with the phorbol ester  did not mimic the effect of bradykinin.Bradykinin can increase glutamate release from mouse astrocytes through volume-sensitive outwardly rectifying (VSOR) anion channels without cell swelling via a mechanism that is regulated by high intracellular Caodomains . Althoug plateau . This ac2+]i responses to bradykinin within a cell population; confocal microscopy studies showed that some cells displayed no plateau phase whereas others were not noticeably affected by bradykinin removal and continued to respond for a few minutes  that enter in a positive feedback loop .2+-dependent transient changes in cultured human fibroblast cytoarchitecture, which share similarities with the increase in cross-sectional area of fibroblasts in response to acupuncture [2+]i signals in cultured human subcutaneous fibroblasts. Controversy still exists regarding adenosine participation in wound healing and scarring. For instance, the adenosine A2A receptor promotes skin fibrosis and scarring i levels in neighboring cells. Thus, targeting the pathways leading to nucleotides release and the purinergic cascade in human fibroblasts of the subcutaneous tissue may be useful in designing novel therapeutic strategies for tuning the communication between inflammatory cells, fibroblasts and sensory nerve endings, which are key players in the pathogenesis of painful musculoskeletal diseases with widespread involvement of the subcutaneous connective tissue .Data indicate that stimulation of constitutively expressed B2PO4, 0.970 g/100 mL K2HPO4 · 3H2O, 0.112 g/100 mL KCl, and 0.084 g/100 mL NaHCO3, pH 7.4) until used, which was between 2 and 16 hours after being harvested i) were measured with the calcium sensitive dye Fluo-4 NW  in a multi detection microplate reader  [2+]i imaging was obtained using a laser scanning confocal microscope i imaging. Cells were cultured for 5-15 days in supplemented DMEM as described before. On the day of the experiment, cells were washed twice with Tyrode’s solution and incubated at 37°C for 45 min with the cell-permeant fluorescent Ca2+ indicator, Fluo-4 NW (2.5 μM), in 1X HBSS, 20 mM HEPES and 2.5 mM probenecid. After removal of the fluorophore loading solution, cells were washed again twice and 150/300 μL of Tyrode’s solution was added per culture well/dish, respectively. The 96-well cell plates were then loaded into the microplate reader. Reader control was performed using the BioTek’s Gen5™ Data Analysis Software. For the recordings, temperature was maintained at 32°C and readings were made with 5 seconds of interval, during approximately 30 minutes, using a tungsten halogen lamp. Fluorescence was excited at 485/20 nm and emission was measured at 528/20 nm. For the single-cell imaging, culture dishes were mounted on a thermostatic (32°C) perfusion chamber at the stage of an inverted laser-scanning confocal microscope equipped with a 20x magnification objective lens . The chamber was continuously superfused (1 mL/min) with gassed  Tyrode’s solution and drugs were delivered using a multichannel valve controlled perfusion system. Changes in fluorescence of the Fluo-4 NW dye were detected in a time-lapse mode. Fluo-4 NW was excited with a 488 nm Multi-line Ar laser and the emitted fluorescence was detected at 510-560 nm, using the scanner of the confocal microscope. Time-lapse sequences were recorded at scanning rates with 20 seconds of interval for approximately 30 min, digitized, and processed off-line by the computer. Regions of interest were defined as bright areas with as little background as possible. For both methods, calcium measurements were calibrated to the maximal calcium load produced by ionomycin . In some experiments, we used rat subcutaneous fibroblasts obtained using the method described before to monitor single-cell [Ca2+]i oscillations (see above) in parallel with the incorporation of FM4-64 , which is a membrane-selective fluorescent dye used to evaluate vesicle endocytosis and exocytosis in living cells  and the slides incubated overnight at 4°C. After incubation, cells were washed 3 times in PBS 1X (10 minutes each). The Alexa Fluor 488 1:1500 (anti-rabbit) and Alexa Fluor 568 1:1500 (anti-mouse) secondary antibodies  were diluted in blocking buffer II  and applied for 1 h protected from light. A last wash was performed with PBS 1X and glass slides were mounted with VectaShield medium and stored at 4°C. For negative controls, the secondary antibodies were applied without pre-incubation with primary antibodies. A positive control for α-smooth muscle actin (SMA) was performed with cardiac myofibroblasts isolated from Wistar rats  using a similar procedure as previously described for human subcutaneous fibroblasts. Observations were performed and analyzed with a laser-scanning confocal microscope -5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridine carboxaldehyde disodium salt (MRS 2211), 2’-deoxy-N6-methyladenosine 3’,5’-bisphosphate tetrasodium salt (MRS 2179), icatibant (HOE-140), R715, sodium metatungstate (POM-1) and thapsigargin were obtained from Tocris Cookson Inc.. Bafilomycin A1 was from WAKO Chemicals and Triton™ X-100 was obtained from Merck. BFA, MFQ, NECA and thapsigargin were prepared in dimethyl sulfoxide (DMSO). All other drugs were prepared in distilled water. All stock solutions were stored as frozen aliquots at -20°C. Dilutions of these stock solutions were made daily and appropriate solvent controls were done. No statistically significant differences between control experiments, made in the absence or in the presence of DMSO at the maximal concentration used (0.05% v/v), were observed. The pH of the solutions did not change by the addition of the drugs in the maximum concentrations applied to the preparations.2-Mercaptoethanol, 2-octanol, 8-phenyltheophylline (8-PT), adenosine 5’-diphosphate sodium salt (ADP), adenosine 5’-monophosphate sodium salt (AMP), adenosine 5’-triphosphate sodium salt (ATP), amphotericin B, apyrase, bovine serum albumin (BSA), bradykinin (BK), brefeldin A (BFA), bromphenol blue, carbenoxolone (CBX), Dulbecco’s Modified Eagle’s Medium (DMEM), ethylene diaminetetraacetic acid (EDTA), ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA), fetal bovine serum (FBS), glycerol, mefloquine (MFQ), 5’-(N-ethylcarboxamide)adenosine (NECA), penicillin/streptomycin, phosphate buffered saline system (PBS), protease inhibitor cocktail, quinacrine dihydrochloride, sodium deoxycholate, sodium dodecyl sulfate (SDS), trypsin, Tween 20, and type I collagenase were purchased from Sigma-Aldrich. n number of experiments/cells/individuals. Data from different individuals were evaluated by one-way analysis of variance (ANOVA) and no significant differences on the pattern of cell behavior were found. Statistical differences found between control and drug-treated cultures were determined by the Bonferroni’s method. p values < 0.05 were considered to represent significant differences.Data are expressed as mean ± S.E.M. from an [Ca2+]i: Intracellular calcium; 8-PT: 8-phenyltheophylline; a.u.: Arbitrary units; ADA: Adenosine deaminase; ADO: Adenosine; AR-C 66096: 2-(propylthio)adenosine-5’-O- triphosphate tetrasodium salt; Baf A1: Bafilomycin A1; BCA: Bicinchoninic acid; BFA: Brefeldin A; BK: Bradykinin; BSA: Bovine serum albumin; CBX: Carbenoxolone; CD39: NTPDase1; CD73: Ecto-5’-nucleotidase; COS-7: Fibroblast-like cell line derived from monkey kidney tissue; Cx: Connexin; DMEM: Dulbecco’s modified eagle’s medium; DMSO: Dimethyl sulfoxide; DRG: Dorsal root ganglia; EDTA: Ethylene diaminetetraacetic acid; EGTA: Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid; FBS: Fetal bovine serum; FITC: Fluorescein isothiocyanate; HBSS: Hank’s balanced salt solution; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HEK-293: Human embryonic kidney 293 cells; HPLC: High-performance liquid chromatography; HRP: Horseradish peroxidase; HX: Hypoxanthine; INO: Inosine; IP3: Inositol trisphophate; MDLK: Madin-Darby canine kidney cells; MFQ: Mefloquine; MRS 2179: 2’-deoxy-N6-methyladenosine 3’,5’-bisphosphate tetrasodium salt; MRS 2211: 2-[(2-chloro-5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridine carboxaldehyde disodium salt; MTT: Thiazolyl blue formazan, 1--3,5-diphenylformazan; NECA: 5’-(N-ethylcarboxamide) adenosine; NTPDase: Nucleoside triphosphate diphosphohydrolase; Panx1: Pannexin-1; PBS: Phosphate buffered saline system; PFA: Paraformaldehyde; POM-1: Sodium metatungstate; PVDF: Polyvinyl difluoride; RLU: Relative luminescence units; SDS: Sodium dodecyl sulfate; SMA: α-smooth muscle actin; TBS: Tris-buffered saline; VSOR: Volume-sensitive outwardly rectifying; WB: Western blot.The authors declare that they have no competing financial interests.All authors contributed to experimental design, execution of lab work and preparation of the manuscript. All authors read and approved the final manuscript.


This review focuses on the Ca2+ control of mechanotransduction and synaptic transmission in sensory hair cells, as well as on Ca2+ signalling in non-sensory cells of the developing cochlea.Calcium ions (Ca Different regions of the cochlear basilar membrane vibrate at different sinusoidal frequencies due to variations in membrane thickness and width from the base (high frequency) to the apex (low frequency) of the cochlea. The basilar membrane supports a polarized sensory epithelium, the organ of Corti, which is responsible for sound transduction; it has the form of an epithelial ridge encompassing highly specialized sensory inner hair cells (IHCs) and outer hair cells (OHCs) characterized by a mechanosensory organelle composed of a stereociliary bundle protruding from the endolymphatic  pole [supporting cells and, in the cochlea, these are flanked by epithelial cells. In the following, supporting and epithelial cells of the sensory epithelium will be collectively designated as cochlear non–sensory cells. In the mature organ of Corti, the supporting cells include inner phalangeal cells, inner and outer pillar cells, outer phalangeal cells , as well as Hensen’s, Böttcher’s and Claudius’ cells. In the lateral direction from the organ of Corti, the epithelium comprises spiral prominence cells and marginal cells of the stria vascularis. Tight junctions between neighboring cells prevent diffusion of proteins between the apical and basolateral domain of hair cells and supporting cells, and insulate endolymph, the unusual extracellular fluid low in Na+ and Ca2+ but rich in K+ that bathes the apical pole of epithelium, from perilymph, the normal extracellular fluid the bathes the basolateral membrane of the cells. In adult wild-type rats, endolymph Ca2+ concentrations have been reported as 20–30 μM [2+ development are not known, it has been reported that a mature composition of endolymph in the mouse cochlea is reached around postnatal (P) day 8  [+ to endolymph and generation of the endocochlear potential  pole   pole ). All cef birth) . The strin refs. -7), an ein refs. .+ are key factors for the mechanotransduction process performed by cochlear hair cells when mechanical stimuli are applied to their stereocilia bundle. Mechanotransduction relies on the large potential difference between the endolymph and the cytoplasm of IHCs and OHCs, which drives K+ through mechanically gated channels in the stereociliary bundle [+ influx through mechanotransduction channels evokes a receptor potential, i.e. a graded change of their resting membrane potential, Vm. This analogue modulation is used by the IHC to activate the synaptic machinery present at its basal pole. By contrast, it is well established that OHCs use their receptor potential to fuel the local mechanical amplification process, driven by the motor protein prestin, which is required for the high sensitivity and sharp frequency selectivity of mammalian hearing , howgreater epithelial ridge, which gives rise to the IHCs and medial non–sensory cells, and the adjacent lesser epithelial ridge, which is thought to give rise to the OHCs and lateral non–sensory cells [epithelial gap junction network forms around embryonic day 16 and connects all supporting cells in the organ of Corti as well as adjacent epithelial cells. A second network, named connective tissue gap junction network, starts to develop around birth and comprises interdental cells and fibrocytes in the spiral limbus, fibrocytes of the spiral ligament, basal and intermediate cells of the stria vascularis (reviewed in refs. [K+recycle (or recirculation) hypothesis . The epin refs. ,20). In in refs. -23), the2+) play numerous and fundamental roles in the inner ear. In the first part of this review, we focus on the aspects of sound transduction that are influenced by Ca2+, including mechanotransduction function and neurotransmitter release at the hair cell synapse. In the second part, we concentrate on Ca2+ signaling in the network of non-sensory cells in the developing cochlea.Calcium ions  and lower (protocadherin 15) part of tip links [2+ binding at interrepeat sites is essential to determine cadherin 23 stiffness and folding strength [2+ binding motif has also been identified at the N terminus of cadherin 23 [In the cochlea, the relative motion between the sensory cells and their overlaying structure, the tectorial membrane, causes the deflection of the hair bundle and the opening of mechanotransduction channels, one of the few ion channels not yet conclusively identified . Stereocng-spring,27, whicependent . It is tependent . Indeed,currents -31. Cadhip links . Furtherstrength . A Ca2+ herin 23 , which cherin 23 ,30. Receherin 23 . Furtherherin 23 -38.2+ essential to preserve the structure of the tip-links, but it also contributes to the mechanotransduction current. Based on early experiments performed under mM levels of extracellular Ca2+ concentration ([Ca2+o), it had been concluded that ~10% of the total mechanotransduction current was carried by Ca2+[2+o in endolymph is as low as a few tens of μM [2+ decrease in proportion to [Ca2+o; at endolympatic levels of [Ca2+o, it accounts for only the ~0.2% of the total mechanotransduction current [0) and the bundle displacement (X) in the direction of the applied stimulus, canceling the effects due to sustained stimuli while maintaining the sensitivity to transient stimuli [2+-dependent, can be distinguished: (i) fast adaptation, which occurs when Ca2+ enters transduction channels, then closes them within a few milliseconds or less; (ii) slow adaptation, which occurs with a time constant that spans a wide range of 10-50 ms depending on the type of hair cells studied .2+ to an intracellular site of the mechanotransduction channel or a closely associated subunit which closes the channel itself [2+-dependent manner with actin filaments at the core of stereocilia [2+ sensitivity of the mechanotransduction channels and the fast adaptation require a structural environment that is dependent on this unconventional myosin; (ii) this environment is disrupted in IHCs of this mutant strain, but not in OHCs. However, available data indicate that myosin-XVa is present at the tips of wild-type stereocilia in both IHCs and OHCs [Fast adaptation is thought to be caused by the direct binding of Cal itself ,44-47. Sl itself , composel itself -50, whicreocilia . The knoreocilia -54. Anotreocilia . This stand OHCs ,57. Thusand OHCs .2+ entry, at the lower end of the tip link [2+ entering through a transduction channel might affect the adaptation motor hooked up to the next tip link lower down the same stereocilium . This i2+ can influence mechanotransduction via cyclic adenosine monophosphate (cAMP), which has been shown to affect the response-displacement curve of the transducer [2+-calmodulin activated type I adenyl cyclase [It is probably worth mentioning also that Caansducer . This si cyclase , cAMP-in cyclase .2+ is rapidly bound by endogenous Ca2+ chelators, present at millimolar concentration [2+ diffuses to a few tens of nm from the mouth of the channel [2+ store [2+ from a system of endoplasmic reticulum membranes located beneath the cuticular plate known as Hensen’s body [After entering the stereocilia through mechanotransduction channels, Cantration , which r channel . Also mi channel ,63, can 2+ store . In OHCs2+ is eventually exported back to endolymph by plasma membrane Ca2+-ATPase (PMCA) pumps, which are highly expressed in the hair bundle of vestibular and cochlear OHCs and, to a lesser extent, IHCs [ATP2b2 gene [w/a splicing isoform of PMCA2 [ATP2b2 gene causes deafness and balance disorders in mice [w/a variant of the pump; they all affected residues that are highly conserved in all PMCA isoforms across species and in other P-type pumps [Tommy mouse mutation was identified as a new PMCA2 pump mutant with progressive deafness from an ENU mutagenesis screen [Cant, IHCs . The stent, IHCs ,66. The 2b2 gene -70. The of PMCA2 ,72. Abla in mice , further in mice ,70,73. Tpe pumps ,74,75. Rs screen . These m2+ at the endolymphatic pole of the hair cell for the performance of the mechanotransduction channel, a diminished Ca2+ removal from the stereocilia is expected to affect the mechanotransduction currents. Indeed, pharmacological blockade [2+ is exported from the stereocilia , its concentration in the endolymph is expected to fall [Due to the crucial role of Cablockade , as wellblockade  have beeblockade ,78. Thus to fall ,79,80.2+-driven action potentials [2+-activated K+ current [K,n, carried by KCNQ4 channels [As mentioned in the introduction, mature hair cells respond to hair bundle deflection with graded changes in their membrane potential, which ultimately result in neurotransmitter release from the cell synaptic pole  clustered at each active zone [2+-activated K+ channels (BK channels) [2+ current (>90%) is carried by CaV1.3 channels [V1.3 channels for ubiquitination and may thus constrain the number of presynaptic CaV1.3 channels in IHCs [V1.3 channels activate at relatively hyperpolarized membrane potential, as negative as -70 mV in immature IHCs [V1.3 channels located within a few nanometer from the release site and such “nanodomain control” of neurotransmitter has been proposed to permit temporally precise synaptic coding even for weak stimuli [Vesicle exocytosis is triggered by the influx of Caive zone -102 (~80ive zone ), where,hannels) . In mammchannels ,104. It channels ,106, tag in IHCs . The bioure IHCs ,108, indure IHCs . Second,ure IHCs ,110, a c stimuli .V1.3 channels show strong Ca2+-dependent inactivation when studied in heterologous expression systems [2+ binding protein (CaBP), which are expressed within the organ of Corti, may moderate the inactivation in cochlear IHCs by competing with calmodulin binding to the channel’s C-terminus [2+- and voltage-dependent inactivation of CaV1.3 channels in IHCs [Because Ca systems , it has terminus ,113. Rec in IHCs .2+, presynaptic Ca2+ domains are presumably spatiotemporally restricted by the presence of mobile, proteinaceous Ca2+ buffers calretinin, calbindin and parvalbumin, which have been found in a variety of cochlear and vestibular hair cells with concentration in the mM range [2+ buffers are saturated, thus permitting fast and large Ca2+ rises near release sites beneath the synaptic ribbon [Similarly to stereociliary CamM range ,115. HowmM range . It has c ribbon .2+ current and transmitter release has been investigated by measuring the increase in the hair cell membrane capacitance (ΔCm) following depolarization-triggered Ca2+ entry [2+ channels [The stimulus-secretion coupling between the inward Ca2+ entry ,117-120.2+ entry . The faschannels -123. Howchannels .2+ influx , at least in high frequency IHCs [rd-5th power) Ca2+-dependance when the hair cell is depolarized to positive holding potentials [2+ uncaging (7 μM to 110 μM) [2+-dependent vesicle trafficking is responsible for this movement, which is required for hair cell synapses to maintain high rates of sustained vesicle fusion [Transmitter release evoked by membrane depolarization over the physiological voltage range  shows ancy IHCs ,120,124.ncy IHCs ,124, is  110 μM) . Recentle fusion .2+ sensor. Synaptotagmins (Syt) I-II are the conventional Ca2+-sensing proteins at neuronal synapses [-/-) are profoundly deaf [2+ sensor of synaptic vesicle fusion in cochlear hair cells [2+ sensor for exocytosis remains indetermined. Indeed, otoferlin is not found in IHCs of a hypothyroid rat model, even though those IHCs exhibited Ca2+-dependent exocytois [2+-evoked exocytosis in the first postnatal days (P0-P4) is both otoferlin- and Syt- independent [2+-dependance observed before and after the onset of hearing doesn’t correlate with the qualitatively similar distribution of otoferlin found in immature and mature IHCs [The identification of the molecular composition of the synaptic machinery of the hair cell remains a major challenge. The hair cell synapse lacks the most common protein involved in exocytosis, for example complexins, synapsins and synaptophysins -128; morsynapses , but thesynapses , more resynapses -133. Howsynapses ,132, Sytdly deaf , and shodly deaf  promptedir cells ,136. Howir cells , its rolxocytois . Moreoveependent . Finallyure IHCs ,139, and2+ influx through voltage-gated Ca2+ channels of the basolateral plasma membrane, two other mechanisms, both implicated in the efferent control of hair cell function [2+ concentration ([Ca2+i) at the basal pole of the hair cell.Besides Cafunction ,141, may2+ entry through α9α10 nicotinic acetylcholine receptors (nAChR) [2+-dependent decrease of axial stiffness in OHCs [The first mechanism is Ca (nAChR) ,143, whi (nAChR) ). The hy (nAChR) -147 and  in OHCs ,149.2+i elevation induces Ca2+ release from internal stores through channels such as inositol-1,4,5-trisphosphate (IP3) receptors (IP3Rs) or ryanodine receptors (RyRs) [2+ release from intracellular store has been found to modulate the fast outward Ca2+ activated K+ current (BK) [The second (interrelated) mechanism is calcium-induced calcium release (CICR), an autocatalytic mechanism whereby [Cas (RyRs) . CICR has (RyRs) , OHCs [1s (RyRs) ,152-156 s (RyRs) . In partent (BK) ,158, thuGJB2 and GJB6 . Thin refs. ). Connexin refs. -166. To in refs. , whereasin refs. .+ recycle is challenged by the identification of connexin26 human recessive deafness mutants, e.g. V84L [+ as the wild type channels, whereas it is the transfer of the Ca2+–mobilizing second messenger IP3 (and possibly of other key metabolites too) that is impaired [+ flux through the hair cells dominates the supply of K+ to the stria vascularis to form a closed K+ cycle see refs. [Mouse models confirmed that connexin26 and connexin30 are essential for auditory function, as well as survival and development of the organ of Corti -175. Tho.g. V84L , that ar.g. V84L . Studiesimpaired ,180. Theimpaired ,182, andee refs. ,184.2 and P2Y4 receptors, also expressed on the endolymphatic surface of the developing sensory epithelium, activates phospholipase–C dependent generation of IP3[3 diffusion through these coupled cells, IP3 binding to its receptors (IP3R) promotes Ca2+ release from the endoplasmic reticulum raising the cytosolic free Ca2+ concentration ([Ca2+i). The probability of connexin hemichannel opening is a bell–shaped function of the [Ca2+i, peaking at ~500 nM [2+ signals as regenerative and coordinated intercellular Ca2+ waves, with peak amplitude of ~500 nM, sustained by ATP–induced ATP–release [2+ buffers and play a significant role in regulating the spatio–temporal properties of these intercellular Ca2+ waves [2+ uptake by dissipating the mitochondrial membrane potential using the protonophore carbonyl cyanide m–chlorophenylhydrazone (CCCP) and oligomycin, an inhibitor of oxidative phosphorylation, or using Ru360, an inhibitor of the mitochondrial Ca2+ uniporter, which enhanced the peak amplitude and duration of ATP–induced transients. The numerous roles played by extracellular ATP in the adult cochlea are reviewed in ref. [While the exact function of connexins expressed by non–sensory cells of the inner ear remains unclear, it is important to mention that they also form unpaired connexons, i.e. non−junctional connexin hemichannels -187. Expon of IP3,193,194. ~500 nM . This is–release ,193,194.2+ waves . This wa in ref. ; the res2+i have been found in a variety of cells and shown to arise spontaneously or after stimulation by hormones or neurotrasmitters. In non–sensory cells of the lesser epithelial ridge, ATP–dependent [Ca2+i oscillations occur (i) as consequence of intercellular Ca2+ wave propagation, (ii) sustained ATP delivery in the submicromolar range or (iii) during pharmacological inhibition of ectonucleotidases, a manipulation which highlights the tonic release of ATP from these cells [2+i transients due to release of ATP in rhythmic bursts have been reported also for a class of non–sensory cells of the greater epithelial ridge (first described by Kölliker) which transiently populate the sensory epithelium from spiral limbus to IHC [2+ signals can be blocked by apyrase, as shown for the propagation of intercellular Ca2+ waves in the lesser epithelial ridge. Furthermore, the frequency of spontaneous [Ca2+i transients is significantly decreased by purinergic receptor antagonists PPADS (50 μM) and suramin (150 μM), the gap junction channel inhibitor carbenoxolone (100 μM) as well as flufenamic acid (50 μM), a bona–fide inhibitor of connexin hemichannels. Both the propagation range of intercellular Ca2+ waves in the lesser epithelial ridge and the frequency of spontaneous [Ca2+i transient in the greater epithelial ridge increase when the extracellular free Ca2+ concentration ([Ca2+o) is decreased [3 precursor in the greater epithelial ridge evokes Ca2+ transients similar to those that arise spontaneously in this region [3 receptor–dependent signal transduction cascades in non–sensory cells of the lesser and the greater epithelial ridge.Rhythms are ubiquitous at all levels of biological organization. At the cellular level, they involve biochemical oscillations that modulate the concentration of key metabolic substrates and second messengers. Among these, rhythmic variations in the [Case cells  and theise cells . In rat se cells ,200, as se cells , [Ca2+i s to IHC ,202. Theecreased ,199,200,ecreased -206. Fins region . Thus it2+ signaling in the developing cochlea. This tenet is exemplified by a study of hearing loss based on the substitution of an evolutionarily conserved threonine by a methionine residue at position 5 near the N–terminus of connexin30 (connexin30 T5M) [T5M/T5M knock in mice, obtained by homologous recombination in mouse embryonic stem cells, expression of the mutated connexin30 T5M protein is under the control of the endogenous connexin30 promoter [T5M/T5M mice exhibit a mild, but significant increase in their hearing thresholds of about 15 dB at all frequencies. Western blot analysis of adult inner ear tissue shows significantly down–regulated expression levels of connexin26 and connexin30. In the developing cochlea, electrical coupling, probed by dual patch–clamp recordings, is normal; however, transfer of the fluorescent tracer calcein between cochlear non–sensory cells is reduced, as is the intercellular Ca2+ signalling due to spontaneous ATP release from connexin hemichannels [2+ oscillations in non–sensory cells of the cochlear feed–back on connexin expression and participate in the coordinated regulation of connexin26 and connexin30 through NF–kB [bovine adeno associated virus (BAAV) vectors restores connexin expression and rescue intercellular coupling and Ca2+ signaling in cochlear organotypic cultures from mice with defective expression of connexin26 and connexin30 [2+i oscillations [2+–dependent transcription factors used by non–excitable cells [These findings are particularly interesting if viewed from the perspective that connexin dysfunction may ensue in a deafness phenotype through a bidirectional link to impaired ATP–dependent Can30 T5M) . In connpromoter . When prchannels . Previough NF–kB ,209 (nucnnexin30 ,209. A wllations ,211, howllations ,213. It llations . Note thle cells , thus fu2+ signaling in the context of the inner ear is a rapidly growing and accelerating field of research. Our knowledge about the molecular details is rapidly growing with new gene products identified as key players of mechanotransduction, while more and more molecules have been added to the list of those involved in synaptic transmission. Finally the interconnectivity of connexin expression and signaling pathways in the developing cochlea constitutes a key feature of the vast non-sensory cell syncitia which starts to emerge as fundamental part of the intricate machinery that fosters the acquisition of hearing. Despite some impressive proceedings in recent years, we are surely not running out of open questions.The investigation of CaThe authors declare no competing interests.FM and FC wrote the paper. Both authors read and approved the final manuscript.


Interestingly, these Ca2+ signals begin most often at the plasma membrane space so that understanding subplasma membrane signals is central to an appreciation of local signaling. Several experimental procedures have been developed to study Ca2+ signals near the plasma membrane, but probably the most prevalent involve the use of fluorescent Ca2+ indicators and fall into two general approaches. In the first, the Ca2+ indicators themselves are specifically targeted to the subplasma membrane space to measure Ca2+ only there. Alternatively, the indicators are allowed to be dispersed throughout the cytoplasm, but the fluorescence emanating from the Ca2+ signals at the subplasma membrane space is selectively measured using high resolution imaging procedures. Although the targeted indicators offer an immediate appeal because of selectivity and ease of use, their limited dynamic range and slow response to changes in Ca2+ are a shortcoming. Use of targeted indicators is also largely restricted to cultured cells. High resolution imaging applied with rapidly responding small molecule Ca2+ indicators can be used in all cells and offers significant improvements in dynamic range and speed of response of the indicator. The approach is technically difficult, however, and realistic calibration of signals is not possible. In this review, a brief overview of local subplasma membrane Ca2+ signals and methods for their measurement is provided. © 2012 IUBMB IUBMB Life, 64(7): 573–585, 2012Ca A fundamental element of signaling is the controlled entry of the ion into the cytoplasm via channels that are often specific for Ca2+ and which respond to extracellular stimulation. This channel activity generates [Ca2+] increases of various time courses and locations that are translated to physiological responses. For example, exocytosis is triggered within microseconds by locally high [Ca2+] near vesicle release sites , gene expression is regulated in minutes . The influx generates a significant local concentration gradient near the channel in which [Ca2+] declines from ∼10 μM to ∼100 nM over a few hundred nanometers from the plasma membrane  to amplify the local signals or propagate through the cell as global signals with slower but more widespread effects throughout the cell  or on (voltage-dependent Ca2+ channels) the plasma membrane  to the plasma membrane in SH-SY5Y cells  via “cAMP junctions” to sensitize IP3R2 to IP3 , to selectively target particular physiological responses, hence the interest in understanding subplasma membrane Ca2+ signaling.An understanding of Camembrane –30. For 5Y cells –37. The ith IP3R –40 that IP3R ]. The clustering enables uniform cell wide activation via depolarization to locally activate selected specific cell processes. In vascular smooth muscle, voltage-dependent Ca2+ channels were suggested to be clustered and constitutively active , a feature which will enable the channels to remain under physiological control , 472+ ch2+]. Some approaches have been based on the sensitivities and activities of Ca2+- activated ion channels, such as the Ca2+-activated K+ channel (KCa) to estimate subplasma membrane Ca2+ concentrations. Using this approach, concentrations of the order of ∼10 μM have been estimated to occur in the subplasma membrane space   rose by only a few hundred nanomolar  to specifically localize Ca2+ indicators to the subplasma membrane space to measure [Ca2+] only there or (2) to allow indicators to be dispersed throughout the cytoplasm but to measure the fluorescence emanating from the Ca2+ signals only at the subplasma membrane space using high resolution imaging procedures. These procedures will now be briefly reviewed.The growing appreciation of the significance of local signals near the plasma membrane in controlling cell function has led to the development of various methodologies to study subplasma membrane  on the inner surfaceof the plasma membrane of the A7r5 smooth muscle cell line to be ∼1–2 μM at rest  was tethered to Na+ pumps to report subplasma membrane Ca2+ signals. Although the indicator reported changes over timescales of minutes (2+ indictors such as fluo-3 (kon of 28.2 μM−1 s−1 and koff of 7.04 s−1 (−1 s−1 and 369 s−1 for fluo-3 (2+ change is 35.5 ms (TN-XL) and 0.14 ms (fluo-3)). The mean capture times were calculated from 1/KonB (2+ binding sites on the indicator and was taken to be 1 μM for each indicator. As shown in 2+] arising from the opening of a single Ca2+ channel declines within 10 ms. Since the mean capture time of the “fast” troponin-based Ca2+ indicator (TN-XL) is 35.5 ms, this probe would fail to detect the Ca2+ change. Fluo-3 is on the other hand fast enough to detect the Ca2+ signal.In cultured cells, the most prevalent approach to measure Ca at rest . The higto 50 μM . Althougameleons” offered  minutes , it was  minutes . Even th7.04 s−1  comparedr fluo-3 . The meam 1/KonB , 57, whe2+ indicator proteins typically have a fairly restricted dynamic range  but results have not been universally positive. For example, rhod-2 has been used in some studies to successfully measure mitochondrial matrix [Ca2+]. Rhod-2 in its membrane permeant acetoxymethyl (AM) ester form bears a net positive charge which, it is suggested, results in mitochondrial accumulation of the dye because of the mitochondria's negative membrane potential. However, rhod-2 bears one net positive charge only in the AM form. When fully de-esterified, rhod-2 bears three net negative charges which is likely to preclude significant mitochondrial accumulation. The balance of esterase activity in the cytoplasm and mitochondria will determine the major source of the signal from rhod-2 and is likely to contribute to variations in results among studies.Although small synthetic molecule Ca2+ indicators in measuring [Ca2+]. Several attempts have been made to target these indicators. In one proof of concept study, peptides which carry organelle-specific import or retention sequences ] has been synthesized that specifically labels fusion proteins that possess four cysteines (tetracysteine tag)  was localized in cells engineered to express SNAP-tag fusion proteins in the nucleus (2+ indicator (a derivative of fura-2 with lower affinity for Ca2+ and faster kinetics) also with a O6-benzylguanine side group has been synthesized to permit subcellular localization again via links to SNAP-tag fusion proteins (kon in the 1–10 × 108 M−1 s−1 range) and high dynamic range (20–100 fold increase in fluorescence from Ca2+-free to Ca2+-bound) of the small synthetic indicators and await full experimental exploitation.If the small molecule indicators could be selectively targeted to subcellular regions, their speed and wide dynamic range could offer significant advantages over fluorescent protein Caequences  were addequences . Althougine tag) . This in signals . In yet  nucleus . A fura-proteins . These p2+ indicator to specific regions of the cell, the other major approach to selectively measure Ca2+ signals from only certain regions has been to use high resolution imaging methods. Indeed, the desire to image Ca2+ at subcellular resolution has been a driving force, which led to significant advances in optics and technology to overcome the constraints imposed on optical microscopy. The main physical limitation imposed by the use of light for high resolution imaging is that of diffraction, which was first fully explained by Ernst Abbe in 1873   , 65 or ay (TIRF) . In TIRFTotal internal reflection occurs when light traveling in a material of high refractive index meets a material of lower refractive index . If the light hits the interface at normal incidence (zero degrees), it will pass through undeviated A. As theAlthough the light is reflected according to Maxwell's equations (which govern the propagation of light) , an elecTIRF, thus, provides a method of selectively exciting molecules close to an interface, and two standard methods have been developed to enable this form of imaging. In the first, a prism is used to direct the light into a glass substrate A. A coll2+ signals and comparison of bulk average [Ca2+]c so that an understanding to the initiation and propagation of the signals is possible.An alternative method, and that used by nearly all commercial TIRF microscopes, is the use of a high numerical aperture objective in an epi-illumination configuration B. In bas00 output (single spot beam with no mode patterns) was coupled using a home built system into a single-mode optical fiber with a numerical aperture (N.A.) of 0.1 and a core diameter of 3 μm. The laser excitation had ∼1.5 mW of light emerging from the end of the optical fiber. The fiber was connected into the Nikon TIRF attachment at the rear of the TE2000, and the laser excitation was directed to the objective by a custom designed dichroic mirror present in the top turret  coupled via a liquid light guide. The monochromator light was directed to the objective by a custom dichroic on the lower epi-illuminator A. This lRapid (2 ms) switching between TIRF and wide-field illumination was achieved by triggering the laser shutter open using a TTL pulse which simultaneously drove the monochomator to 250 nm . Terminating the TTL pulse shuttered the laser closed and moved the monochromator to 490 nm.−1) or Cascade 512B (∼30 frames s−1)) (One unexpected issue arose from the movement of the monochomator between 250 nm and 470 nm. Although the transition only took 2 ms when compared to the CCD cameras frame switch (∼2 μs) in frame transfer mode, it was relatively slow. As a result, a small amount of light initially was recorded during the 2 ms monochromator switch before the final desired wavelength was reached. The light recorded during the monochromator wavelength switch created a slightly increased background fluorescence which limited the ability to resolve of small signals. After exploring several possibilities, the simplest solution was to add a 490 nm narrow bandpass excitation filter to the monochromator excitation light path. Imaging was achieved using EMCCD camera's (a Photometrics Evolve 128 (∼300 frames ses s−1)) A.2+ signals measured in the evanescent field are the sum of signals ranging from saturated fluorophore (near to the channel) to infrequent binding events . Fluorophore saturation will mean the gradient cannot easily be deconvolved so that the fluorescent signal is likely to be an especially nonlinear representation of [Ca2+] changes. Even if the dye did not saturate, converting the fluorescent signal to a Ca2+ concentration relies on calibration carried out in steady-state conditions. Such conditions are unlikely to be valid in the rapidly changing microdomains under in the subplasma membrane space during Ca2+ influx. Indeed it is likely that the assumption that the dye equilibrates instantly with Ca2+ will not be correct during influx near a Ca2+ channel which is rapid (∼0.6 million ions per second), that is, there is local nonequilibrium within the system (2+ indicators are too slow to follow these fast [Ca2+] increases. Notwithstanding these difficulties, TIRF is a powerful method to study subplasma membrane events and has generated significant new insights to channel behavior and Ca2+ signaling.One difficulty which arises when using freshly isolated smooth muscle cells comes from their crenulated structure, which may result in the cells not lying completely flat on the coverslip. The evanescent wave extends only ∼100 nm from the coverslip C and if e system . Even th2+ signals may be highly localized and extremely rapid events that have a timecourse approximately four orders of magnitude faster than changes in bulk average [Ca2+]c (2+] changes with the speed of the effectors Ca2+ binding. Local subplasma membrane Ca2+ signals have been studied using targeted Ca2+ indicators but the limited dynamic range and slow reaction to changes in [Ca2+] may not fully describe the response. Rapid small molecule Ca2+ indicators used with high resolution imaging have significantly improved dynamic range and speed but the approach is technically difficult and realistic calibration of signals is not possible. Synthetic indicators that are targeted to particular subcellular sites would combine specificity of the protein biosensors approach with the fast kinetics and high dynamic range of small molecular indicators; these are under development and await full experimental exploitation.Subplasma membrane Ca [Ca2+]c . These s


Here we provide evidence that these actions of GLP-1 are functionally related: they reflect a process of Ca2+-induced Ca2+ release (CICR) that requires activation of protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs). In rat insulin-secreting INS-1 cells or mouse β cells loaded with caged Ca2+ (NP-EGTA), a GLP-1 receptor agonist (exendin-4) is demonstrated to sensitize intracellular Ca2+ release channels to stimulatory effects of cytosolic Ca2+, thereby allowing CICR to be generated by the uncaging of Ca2+ (UV flash photolysis). This sensitizing action of exendin-4 is diminished by an inhibitor of PKA (H-89) or by overexpression of dominant negative Epac. It is reproduced by cell-permeant cAMP analogues that activate PKA (6-Bnz-cAMP) or Epac (8-pCPT-2′-O-Me-cAMP) selectively. Depletion of Ca2+ stores with thapsigargin abolishes CICR, while inhibitors of Ca2+ release channels (ryanodine and heparin) attenuate CICR in an additive manner. Because the uncaging of Ca2+ fails to stimulate CICR in the absence of cAMP-elevating agents, it is concluded that there exists in β cells a process of second messenger coincidence detection, whereby intracellular Ca2+ release channels  receptors) monitor a simultaneous increase of cAMP and Ca2+ concentrations. We propose that second messenger coincidence detection of this type may explain how GLP-1 interacts with β cell glucose metabolism to stimulate insulin secretion.The blood glucose-lowering hormone glucagon-like peptide-1 (GLP-1) stimulates cAMP production, promotes Ca Insulined fully .S GTP-binding proteins, but also by Ca2+/calmodulin  receptors, IP3-R), the opening of which is reported to be facilitated by Ca2+ and cAMP (2+ through voltage-dependent Ca2+ channels (VDCCs), it is predicted that GLP-1 and glucose should interact synergistically to gate Ca2+ release channels from a closed to open state. Indeed, second messenger coincidence detection of this type might explain the unusual interaction of GLP-1 and glucose to mobilize an intracellular source of Ca2+ in the β cell  mediated by the RYR and IP3-R. Sensitization allows CICR to be triggered by the uncaging of Ca2+ in INS-1 cells or mouse β cells loaded with a photolabile Ca2+ chelator , and the Epac family of cAMP-regulated guanine nucleotide exchange factors . The mice were anaesthetized by inhalation of CO2 , and were killed by cervical dislocation. Surgical procedures for removal of the pancreas were performed in accordance with NYU School of Medicine policies governing the ethical use of mice for experimentation (IACUC Protocol no. 040602-01). After digestion of the pancreas with collagenase P , batches of 150–200 islets were subjected to mild trypsinization and were dispersed by trituration in a Ca2+-free saline in order to generate a single cell suspension. Isolated cells were then allowed to adhere to glass coverslips  coated with concanavalin A . Primary cultures were maintained in a humidified incubator  at 37°C in RPMI 1640 supplemented with 10% FBS, 100 units ml−1 penicillin G, and 100 μg ml−1 streptomycin. β cells were identified on the basis of their large diameter and granular appearance. INS-1 cells (passage numbers 70–90) were maintained in RPMI 1640 containing 10 mm Hepes, 11.1 mm glucose, 10% FBS, 100 units ml−1 penicillin G, 100 μg ml−1 streptomycin, 2.0 mml-glutamine, 1.0 mm sodium pyruvate, and 50 μm 2-mercaptoethanol : 138 NaCl, 5.6 KCl, 2.6 CaCl2, 1.2 MgCl2, 10 Hepes, 11.1 d–glucose and supplemented with 1 μm fura−2 AM , 2% FBS, and 0.02% Pluronic F-127 . Cells were exposed to the fura−2 loading solution for 20–30 min at 22°C. Experiments were performed in SES at 32°C using a TE300 inverted microscope  equipped with a temperature-controlled stage  and a 100× Nikon UVF oil immersion objective (NA 1.3). Microfluorimetry was performed ratiometrically at 0.5 s intervals using a video imaging system outfitted with an intensified CCD camera . A rotating mirror delivered excitation light at 340 or 380 nm. The emitted light was measured at 510 nm, and the average of 29 frames of imaging data was used to calculate numerator and denominator values for determination of 340/380 ratios after background subtraction. [Ca2+]i was calculated according to established methods . Values of Rmin and Rmax were 0.20 and 7.70. Components of the imaging system are illustrated  containing  or caged IP3 . The loading solution also contained 1 μm fura-2-AM, 2% FBS, and 0.02% Pluronic F-127. ci-IP3/PM is a photolabile caged IP3 related in structure to cm-IP3/PM . In ci-IP3/PM, 2- and 3-hydroxyl groups of myo-inositol are protected by an isopropylidene group; whereas in cm-IP3/PM, 2- and 3-hydroxyl groups are protected by a methoxymethylene group. Like cm-IP3/PM, ci-IP3/PM is cell permeable, and once inside cells, induces Ca2+ release from intracellular stores upon UV flash photolysis : 138 NaCl, 5.6 KCl, 2.6 CaCl2, 1.2 MgCl2, 25 NaHCO3, 10 Hepes-NaOH (pH 7.40), and 11.1 glucose (INS-1 cells were transiently transfected with a plasmid encoding yellow cameleon 3.3-er (YC3.3-er) ( glucose .m fluo-4 AM (Molecular Probes). The [Ca2+]i was imaged using a laser scanning confocal microscope  equipped with a 63× water immersion objective (NA 1.2). Fluo-4 was excited at 488 nm using an argon laser. The emitted light passed through a 500 nm dichroic filter for detection using a fluo-4 emission filter . Images of the x–y optical sections were recorded with a resolution of 512 pixels line−1 at 400 Hz. Raster point size was 0.1 μm, with an overall lateral resolution of 0.16 μm. For each data set, 20 x–y scans of a small cluster of cells were acquired. After the third section was scanned, a test solution was applied via a micropipette. Image analysis was performed using Leica Confocal Software .Cells plated on 0.15 mm glass coverslips were incubated for 20 min at room temperature in SES containing 5 μm): 110 KCl, 10 NaCl, 2 MgCl2, 20 Hepes, 5 KH2PO . The heparin was injected from Femtotip II needles into individual β cells using a Transjector 5246 microinjection system  mounted on a Nikon TE200 inverted microscope. The injection pressure was 100 hPa, the compensation pressure was 33 hPa, and the duration of injection was 0.2 s. In order to identify β cells injected with heparin, the injection solution also contained fluorescein . The injected cells were then loaded with fura 2 AM and caged compounds. Prior to imaging of Ca2+, cells injected with heparin and fluorescein were visualized using an EYFP filter set.Low molecular weight heparin  was dissolved in buffer containing (mEpac1 (GenBank Accession No. AAF103905) (Epac1 (R279E) in pcDNA3.1 were obtained from Dr X. Cheng   and dominant negative Epac2  in pSRα were provided by Dr S. Seino   to insert the FLAG epitope at the exchange factor's N-terminus. Epac constructs were introduced into INS-1 cells using LipofectAMINE Plus (Invitrogen-Life Tech.). Cells transfected with wild-type or dominant negative Epac were identified by cotransfection with pEYFP-N1 . A 1: 4 molar ratio of pEYFP-N1 relative to Epac plasmid was used in all transfections .Human wild-type Whole cell lysates of transfected INS-1 cells expressing recombinant Epac were dissolved in 1× Laemmli's sample buffer, boiled for 5 min, centrifuged to remove unsolubilized material, and resolved by SDS-PAGE using 4% stacking and 12% resolving gels. The resolved Epac proteins were transferred to Immobilon-P PVDF membrane  by electrophoretic transfer . Western immunoblot analyses were performed using mouse anti-FLAG monoclonal primary antiserum  in combination with goat antimouse polyclonal secondary antiserum (1: 5000 dilution) conjugated to horseradish peroxidase (Sigma-Aldrich).Luciferase activity was measured in lysates of INS-1 cells transfected with CRE-Luc  as described . This coO-Me-cAMP were from BioLog Life Science . Test solutions dissolved in SES were added to the bath solution or were applied to individual cells from glass ‘puffer’ micropipettes  using a pressure ejection system  as described . 8-pCPT-cAMP, 6-Bnz-cAMP, and 8-pCPT-2′-−1 were selected one at a time in random order to determine basal [Ca2+]i and CICR amplitude. CICR in INS-1 cells was defined as a transient increase of [Ca2+]i, the duration of which did not exceed 30 s when measured at the 10% amplitude cut-off  cut-off C. An add cut-off C. Becaus2+ from NP-EGTA, a photolabile Ca2+ chelator. Prior to the loading of cells with NP-EGTA, the resting [Ca2+]i was 135 ± 65 nm . After loading with NP-EGTA, there was a statistically significant reduction of [Ca2+]i to 91 ± 5 nm . A 600 μsec flash of UV light produced a small increase of [Ca2+]i in these cells loaded with NP-EGTA  . It may be concluded that the increase of [Ca2+]i depicted in 2+.To assess the properties of CICR in INS-1 cells, we performed UV flash photolysis to uncage Ca0 cells) A. This ipeatable A. No sucm) was applied to single INS-1 cells loaded with NP-EGTA, no increase of [Ca2+]i was measured (2+]i (CICR) was observed when Ex-4 was applied simultaneous with the uncaging of Ca2+ . Sensitization of CICR was also observed when UV flash photolysis was performed in the presence of cAMP-elevating agent forskolin (2 μm)  D. This a2+ may interact to generate CICR in INS-1 cells. To evaluate this possibility, we examined whether cell-permeant cAMP analogues sensitize the CICR mechanism of INS-1 cells in a manner analogous to that described for Ex-4 and forskolin. When NP-EGTA-loaded INS-1 cells were exposed to 8-pCPT-cAMP (100 μm), an activator of both PKA and Epac, the uncaging of Ca2+-triggered CICR (m), a PKA inhibitor (O-Me-cAMP (100 μm), a cAMP analogue active at Epac only (m), an analogue active at PKA only  was without effect, a higher concentration (10 μm) reduced the percentage of cells responding to Ex-4 by 60%  reduced the percentage of INS-1 cells exhibiting CICR under conditions of UV flash photolysis. This was the case when the uncaging of Ca2+ was performed in the presence of Ex-4 (1 nm) or 8-pCPT-cAMP (100 μm) ((1 mm) mimicked the action of Ex-4  and forskolin (2 μm) allowed for the appearance of CICR  (A 10–15 min pretreatment of INS-1 cells with ryanodine (10 μ(100 μm) A. Caffei of Ex-4 C, and th of Ex-4 A. To val of CICR A and B. n (1 μm) C and D.m) produced a transient increase of [Ca2+]i in INS-1 cells not loaded with NP-EGTA. This action of caffeine was fast in onset and recovered to baseline within 10–20 s , as demonstrated using INS-1 cells expressing a cameleon Ca2+ reporter (YC3.3-er) targeted to the ER. When 10 mm caffeine was administered to these cells, a transient decrease of 535/485 nm emission ratio was detected (2+]ER) . This value decreased to 87 ± 14 nm (n= 22 cells) in NP-EGTA-loaded cells . The uncaging of Ca2+ in β cells generated a small increase of [Ca2+]i but did not initiate CICR . In these same cells, application of Ex-4 (1 nm), forskolin (2 μm), or the Epac-selective cAMP analogue 8-pCPT-2′-O-Me-cAMP (100 μm) failed to alter resting [Ca2+]i (O-Me-cAMP (100 μm) was highly effective in this assay. It allowed for the appearance of CICR in 8 of 15 cells tested. Similar to findings obtained with INS-1 cells, no sensitization of CICR by forskolin was observed when mouse β cells were treated with thapsigargin  rendered forskolin less effective in the assay of β cell CICR reported here (m), a sensitizer of RYR, allowed for the appearance of CICR under conditions of UV flash photolysis. This action of caffeine was also diminished by ryanodine . The uncaging of IP3 generated a small increase of [Ca2+]i under conditions of low-intensity UV flash photolysis, but it did not trigger CICR (m), the small increase of [Ca2+]i was converted into a large Ca2+ spike  of secretory granules in β cells (2+]i increases to low micromolar levels. Since the mechanism of cAMP-dependent Ca2+ mobilization reported here generates an increase of [Ca2+]i in the 0.5–2 μm range, it may serve as an adequate stimulus for exocytosis of the HCSP.Prior studies demonstrate that cAMP-elevating agents mobilize Ca insulin b. Thus,  β cells . These g2+ and UV flash photolysis to produce a small increase of [Ca2+]i. Under these conditions, the uncaging of Ca2+ triggers CICR when Ca2+ release channels are sensitized by cAMP-elevating agents. Of particular note is our demonstration that the uncaging of Ca2+ fails to generate CICR in the absence of cAMP-elevating agents. This key observation leads us to conclude that a simultaneous increase of intracellular Ca2+ and cAMP concentrations is a necessary prerequisite for the initiation of CICR in β cells. Fundamentally, this is a process of second messenger coincidence detection, and it results from dual stimulatory actions of Ca2+ and cAMP at Ca2+ release channels. These Ca2+ release channels correspond to RYR and the IP3-R, as demonstrated by antagonism of CICR following application of ryanodine or heparin.Here we describe an experimental strategy by which the existence of cAMP-regulated CICR in β cells may be validated. The strategy relies on the use of caged Ca3-R-mediated Ca2+ mobilization is also demonstrated through our use of ci-IP3/PM, a membrane-permeant caged IP3 that activates the IP3-R in a selective manner. Under conditions in which β cells are treated with forskolin, the uncaging of IP3 stimulates CICR. This action of forskolin might reflect cAMP-dependent sensitization of the IP3-R. Alternatively, cAMP may sensitize RYR to stimulatory effects of Ca2+ released from activated IP3 receptors. If so, heparin would act at the IP3-R to prevent the small rise of [Ca2+]i that acts as an initiator of CICR mediated by RYR. Indeed, the mobilization of Ca2+ by IP3 in pancreatic acinar cells is reported to trigger CICR mediated by ryanodine receptors (3 receptors and RYR in β cells.IP2+ stores (2+]i (2+ stores are full and the cytosolic Ca2+ concentration is elevated sufficiently to allow CICR to be generated when Ca2+ release channels are sensitized by cAMP. Context specificity of this type is likely to be of physiological significance, because it may explain, at least in part, why the insulin secretagogue action of GLP-1 is strictly dependent on exposure of β cells to glucose (Prior studies of β cells demonstrate that CICR occurs in a context-specific manner (es (2+]i . Therefo glucose .2+-mobilizing action of Ex-4 reported here. This action of caffeine is inhibited by ryanodine, thereby confirming the existence of CICR mediated by RYR. Such findings are consistent with prior studies demonstrating ryanodine and caffeine-sensitive mobilization of Ca2+ in β cells or β cell lines (2+ mobilized may include the ER because we find that caffeine and forskolin fail to promote CICR in thapsigargin-treated cells. Moreover, caffeine releases ER Ca2+, as measured in INS-1 cells expressing YC3.3-er.Because insulin-secreting cells express RYR, albeit at low levels (ll lines . The sou2+]i, exocytosis, apoptosis, and endosome function in β cells (2+ influx (2+]i in response to a low concentration of ryanodine, thereby demonstrating the presence of RYR in this cell type (2+]i in response to GLP-1, an action inhibited by a high concentration of ryanodine (RYR is implicated in the regulation of [Ca+ influx . In contRYR1 and RYR2 mRNA are expressed in islets (RYR mRNA by RT-PCR of purified preparations of mouse β cells (New studies demonstrate the presence of immunologically detectable RYR in human and mouse β cells (3-R are also of interest. Although activation of the GLP-1-R fails to stimulate IP3 production in islets or INS-1 cells (3-R function. This action of cAMP is PKA mediated (2+ from IP3-R-regulated Ca2+ stores may explain, at least in part, how GLP-1 stimulates exocytosis in β cells (3-R-mediated CICR sensitized by a cAMP-elevating agent and which is blocked by treatment of β cells with heparin, an inhibitor of IP3-R function.Actions of GLP-1 mediated by the IP2+ exerts stimulatory effects on IP3 production in insulin-secreting cells (2+ is most likely a consequence of its ability to activate phospholipase C (2+ were to exist in β cells loaded with NP-EGTA, the uncaging of Ca2+ might raise levels of IP3, thereby stimulating the IP3-R. CICR triggered in this manner might be facilitated under conditions in which the IP3-R is sensitized by cAMP-elevating agents. Because evidence also exists for ‘atypical’ mechanisms of Ca2+ release in the β cell (3-R.It is also important to note that Ca3-R (2+ release channels might require formation of an activated Epac/Rap signalling complex. Such an action of Epac would complement its ability to interact with secretory granule-associated proteins (2+-dependent exocytosis in the β cell (Prior studies of multiple cell types demonstrate that cAMP acts via PKA to sensitize RYR and the IP3-R a. Theref3-R Ca2+ release channels mediate the Ca2+ mobilizing action of GLP-1 in β cells. Both types of Ca2+ release channels act as cAMP and Ca2+ coincidence detectors, because their opening is facilitated by a simultaneous increase of intracellular cAMP and Ca2+ concentrations. PKA and Epac-mediated sensitization of CICR demonstrated here may provide a new explanation for how GLP-1 interacts with β cell glucose metabolism to stimulate insulin secretion.Findings presented here suggest that RYR and IP