Ca2+, acting with calmodulin, can also increase the activity of PDE1-family phosphodiesterases (PDEs), which cleave the phosphodiester bond of cAMP. Surprisingly, SOCE-regulated cAMP signaling has not been studied in cells expressing both Ca2+-sensitive enzymes. Here, we report that depletion of ER Ca2+ activates PDE1C in human arterial smooth muscle cells (HASMCs). Inhibiting the activation of PDE1C reduced the magnitude of both SOCE and subsequent Ca2+/calmodulin–mediated activation of ADCY8 in these cells. Because inhibiting or silencing Ca2+-insensitive PDEs had no such effects, these data identify PDE1C-mediated hydrolysis of cAMP as a novel and important link between SOCE and its activation of ADCY8. Functionally, we showed that PDE1C regulated the formation of leading-edge protrusions in HASMCs, a critical early event in cell migration. Indeed, we found that PDE1C populated the tips of newly forming leading-edge protrusions in polarized HASMCs, and co-localized with ADCY8, the Ca2+ release activated Ca2+ channel subunit, Orai1, the cAMP-effector, protein kinase A, and an A-kinase anchoring protein, AKAP79. Because this polarization could allow PDE1C to control cAMP signaling in a hyper-localized manner, we suggest that PDE1C-selective therapeutic agents could offer increased spatial specificity in HASMCs over agents that regulate cAMP globally in cells. Similarly, such agents could also prove useful in regulating crosstalk between Ca2+/cAMP signaling in other cells in which dysregulated migration contributes to human pathology, including certain cancers.In addition to maintaining cellular ER Ca For instance, the Ca2+-sensitive subgroup of adenylyl cyclases (ADCYs) are uniquely sensitive to SOCE-derived Ca2+ -activated forms of ADCY (2+-inhibited forms of ADCY (2+) store depletion, independently of changes in cytosolic Ca2+  and numerous Ca2+-insensitive cAMP-hydrolyzing PDEs . In contrast, only phenotypically activated HASMCs express a Ca2+/CaM-activated cAMP PDE   in healthy arteries express a “contractile” phenotype. “Contractile” HASMCs are excitable cells with limited proliferative or migratory capacities . In resphenotype . “Activahenotype . Comparee HASMCs . In addid HASMCs , inductihenotype . In addi, PDE1C) , 28, 29.2+/CaM-activated, cAMP-hydrolyzing, PDE  in cells would influence SOCE-mediated changes in cAMP synthesis in cells expressing Ca2+-sensitive ADCYs. Herein, we describe results of experiments aimed at answering each of these important questions. First, we show that depleting ER(Ca2+) stores activates PDE1C. Second, we show that inhibiting this PDE1C activation reduces SOCE as well as SOCE-dependent ADCY8 activation in these cells. Functionally, we show that PDE1C-mediated cAMP hydrolysis, through its impact on SOCE-mediated activation of ADCY8 in HASMCs, controls formation of actin-rich leading-edge protrusions (LEPs) in polarized migrating HASMCs. Taken together, our findings identify a novel signaling system through which it may be possible to promote the healing functions associated with HASMC migration and to limit the maladaptive effects of exaggerated HASMC migration in vascular conditions, including atherosclerosis or poststenting restenosis.To date, few studies have comprehensively investigated the impact of SOCE on cAMP-signaling in HASMCs. Further, no studies have assessed how expression of a CaA). As predicted, decreases in fluorescence resonance energy transfer (FRET)  were recorded in H134-expressing HASMCs incubated with the ADCY activator forskolin (Fsk), and these responses were blocked by the transmembrane ADCY inhibitor, SQ22536 (A and B). ER(Ca2+) store depletion  also decreased FRET   in H134-expressing HASMCs. Indeed, in the 139 cells studied, ER(Ca2+) store depletion increased cAMP by an average of 8.3 ± 0.7% (mean ± SEM). Subsequent SOCE (Krebs + CPA) caused temporally biphasic changes in cAMP in these H134-expressing HASMCs . SOCE was associated with an initial transient decrease in cAMP  which was followed by larger and longer-lived increase in cAMP . Of note, SOCE-associated increases in HASMC cAMP were on average 46.5 ± 1.4% (mean ± SEM) of the maximum increase recorded in these cells [Fsk (10 μM) + 3-isobutyl-1-methylxanthine ] .HASMC cAMP levels were monitored in cells expressing a validated cAMP sensor )  Fig. 1AAFig. 1A. SQ22536 , A and B2+) store depletion . While the initial transient decrease in cAMP associated with SOCE was reduced somewhat by SQ22536, this did not reach significance (C and E). In contrast, SQ22536 did reduce markedly the magnitude and rate of the increase in cAMP observed later during SOCE . To determine whether these effects reflected Ca2+-mediated effects on the activities of the Ca2+-sensitive ADCYs expressed in HASMCs , experiments were also conducted in ADCY6-silenced or ADCY8-silenced HASMCs, respectively. Silencing ADCY6, but not ADCY8, blunted cAMP increases associated with ER(Ca2+) store depletion (compare H and I and M and N). Similarly, SOCE-associated changes in cAMP were also differentially affected by silencing of ADCY6 or ADCY8. Indeed, while silencing either of these ADCYs did significantly reduce the magnitude of the early SOCE-associated decrease in cAMP , only silencing ADCY8 significantly reduced the subsequent SOCE-associated increase in cAMP . Interestingly, although ADCY6 did not reduce the magnitude of the SOCE-associated increase in cAMP, it did reduce the rate at which cAMP accumulated during this phase of the response . Because the silencing efficiency of the ADCYs were similar (R and S), we suggest this did not represent an important factor in the different responses seen in these experiments. Taken together, our data show that ADCY6 activity was involved in regulating changes in HASMC cAMP observed during ER(Ca2+) store depletion and early during SOCE and that ADCY8 activity was dominant in regulating the cAMP increases triggered by SOCE in these cells.Transmembrane ADCY inhibition with SQ22536 did not alter cAMP changes associated with ER(Ca2+) store depletion or in response to SOCE, we monitored PKA activity in cells expressing a PKA activity sensor (AKAR4 FRET sensor (A and B). In these experiments, we observed that PKA activity was elevated during both ER(Ca2+) store depletion and SOCE (B and C), with a larger fractional increase being associated with SOCE (D and E).Previous reports indicated that SOCE-mediated activation of ADCY8 could increase cellular PKA activity , 31. To T sensor ) Fig. S+ store dand SOCE , B and Cith SOCE , D and E2+) store depletion and SOCE each increased cAMP PDE activity in HASMCs (A) and silencing the Ca2+-sensitive cAMP PDE  obviated these effects (B and D). Because silencing PDE1C did not alter Ca2+-insensitive cAMP PDE activity levels in these cells (2+) store depletion, or SOCE, we next studied how its inhibition or silencing altered their associated intracellular Ca2+ transients. For these experiments, PDE1C was inhibited using either one of two structurally distinct PDE1-family selective inhibitors, C33 or PF-04827736  [34–35], and PDE1C was silenced with one of two selective PDE1C-targetting siRNAs  store depletion , B and C (quantitation)). In contrast, PDE1C inhibition did reduce the maximum level of Ca2+ entry detected during SOCE and its rate of entry in HASMCs . Consistent with the idea that PDE1 inhibitors acted specifically on PDE1C, silencing this PDE significantly reduced SOCE in these cells . Because inhibition or silencing of the dominant Ca2+-insensitive PDEs only modestly altered maximal Ca2+ increases during SOCE in HASMCs (A–C), our data identify PDE1C as a selective regulator of cAMP’s actions on SOCE in these cells.ER store depletion did promote formation of Orai-1/Stim-1-containing puncta in HASMCs, silencing PDE1C did not alter their numbers or the expression of the ER luminal Ca2+-sensing protein, Stim-1 (A–D). These findings indicate that PDE1C is unlikely to regulate HASMC SOCE by altering ER(Ca2+) sensing or the dynamics of Orai1-Stim-1 interactions in these cells.Orai1/STIM1-containing microscopic plasma membrane le cells , 34. Whi, Stim-1 , A–D. Th2+) nor the increase caused by ER(Ca2+) store depletion (0 Ca2+ + CPA) , Fig. 4B (quantitation)). In contrast, inhibiting PDE1C reduced the early SOCE-associated decrease in cAMP (A and C) and the rate at which cAMP accumulated during the latter phase of SOCE . Similarly, silencing PDE1C did not alter cAMP increases caused by HASMC ER(Ca2+) store depletion (F and G) but did reduce the magnitude of both the early and late SOCE-associated changes in cAMP (F and H–J). Interestingly and consistent with the idea that PDE1C is unique in its ability to affect SOCE-associated changes in HASMC cAMP, although PDE4 inhibition  significantly increased basal cAMP in H134-expressing HASMCs , the temporally biphasic changes in cAMP associated with SOCE activation were completely unaltered by inclusion of the PDE4 inhibitor in our experiments (A–D). Taken together, these data support the hypothesis that PDE1C activation, perhaps in combination with ADCY6 inhibition, contributes to the early transient decrease in cAMP seen during SOCE. Moreover, they support the idea that inhibiting, or silencing, PDE1C, perhaps by reducing SOCE, decreases SOCE-mediated activation of ADCY8 in these cells. Lastly, given that PDE4 activity accounts for ∼65% of HASMC cAMP PDE activity  in these cells reduced, rather than promoted, formation of these structures (2+-insensitive cAMP PDE activity (PDE4) was inhibited . In contrast, but as predicted based on previous work , C–E) and antagonized the LEP promoting effects of PDE1C inhibition (F and G). Consistent with an important role for PKA in these events, addition of the cell-penetrant PKA inhibitory peptide (Myr-PKI) inhibited LEP formation and antagonized the LEP promoting actions of silencing PDE1C . Addition of a cell-permeant AKAP–PKA disruptor peptide, St-Ht31 (HT31), but not its inactive variant, St-Ht31(P) (pHT31), reversed the LEP promoting effects associated with PDE1C inhibition , indicating that PKA compartmentation within an AKAP-based signalosome is critical in these events. As was the case with another cAMP effector, exchange protein activated by cAMP-1 , reduced basal LEP formation and inhibited the LEP promoting effects of each of the two structurally distinct PDE1-family inhibitors used in our studies . Because our data showed that PDE1C inhibition or silencing of this PDE more markedly impacted ADCY8-associated changes in HASMC cAMP, we next investigated the dependence of this ADCY on LEP formation. Interestingly, although ADCY6-silenced HASMCs produced fewer LEPs than control cells, PDE1C inhibition still promoted LEP formation in these cells . In marked contrast, while silencing ADCY8 did not statistically reduce LEP formation, PDE1C inhibition did not promote formation of these structures in ADCY8-silenced HASMCs ; an effect consistent with PDE1C acting in concert with ADCY8, but not ADCY6, to regulate LEP formation. Together, these findings show that PDE1C likely acts within an AKAP-based, PKA-containing, and perhaps exchange protein activated by cAMP-1–containing signalosome to regulate HASMC LEP formation and that ADCY8 was dominant in providing the source of cAMP that allowed this system to function in these cells.Consistent with our previous work , silencious work , ADCY acy cAMP-1 , silenciA (representative traces), B and C (quantitation)].Because our data linked SOCE and LEP formation in HASMCs and previous reports indicated that greater SOCE occurred at the front of migrating cells , 5, 31, Fig. S8A). Potentially speaking to the selectivity of these interactions, anti-PDE1C immune isolates did not contain gravin, a distinct HASMC-expressed AKAP, STIM-1, or PKARIα . Although overexpressed HA-tagged ADCY8 could sometimes be recovered in anti-PDE1C isolates (not shown), very high levels of background associated with overexpression of this enzyme precluded confirming its presence in these complexes in these experiments.Previous studies, largely using heterologous expression systems, indicated that Orai1, AKAP79, PKA, and ADCY8 could be co-immunoprecipitated from certain cell lysates , 14. BecFig. 6B) and that GFP-STIM1 staining was largely, but not exclusively, perinuclear (B). Interestingly, although FLAG-PDE1C was detected throughout HASMCs, its staining was more abundant in cellular regions devoid of cell–cell contacts . When HASMC LEPs were immunostained, each of the proteins of interest were detected in these structures. Indeed, staining detected each AKAP79, myc-Orai1, and HA-ADCY8 in HASMC LEPs. Interestingly, staining for myc-Orai1 and HA-ADCY8 was relatively more abundant at the tips, than the bulk cytosol of LEPs . Consistent with the idea that ER(Ca2+) release occurs at leading edges of polarized and migrating cells, GFP-STIM1 staining was detected in LEPs . Most strikingly, while levels of FLAG-PDE1C staining was low in the bulk cytosol of HASMC LEPs, this staining was highly concentrated at the tips of these structures ; a result suggesting that PDE1C concentrates at the tips of these structures. Although the dynamic nature of these structures  or Ca2+-dependent, but CaM-independent, inhibition of others (ADCY5 or ADCY6)  . In cont2+/CaM-activated cAMP PDE expressed in HASMCs  was regulated in the context of SOCE and the impact of this regulation on SOCE-ADCY crosstalk in HASMCs, a critical vascular cell type. These issues were studied by inhibiting, or silencing, PDE1C and monitoring how these interventions modified SOCE-dependent and SOCE-independent cAMP homeostasis in HASMCs. Our findings showed that increases in intracellular Ca2+, either resulting from ER(Ca2+) store depletion or SOCE, activated PDE1C and conclude that this PDE1C activation reflects, at least in part, Ca2+/CaM binding and activation. Because PDE1C activation survived cell lysis, we further predict that some PDE1C activation may reflect protein phosphorylation or some other stable modification. Of course, our future work will address the nature of this effect more formally. Importantly, however, we report here that PDE1C activation influenced the magnitude and rate of SOCE-mediated increases in intracellular Ca2+, and likely because of these effects, the ability of SOCE to regulate selectively the activities of each of the Ca2+-sensitive ADCYs expressed in these cells, namely ADCY6 and ADCY8. In relation to their impact on important cellular functions, our finding showed how PDE1C regulated formation of LEPs in polarized HASMCs, a critical early event required for cell migration.Herein, we described results of studies aimed specifically at assessing whether the activity of the CaOur findings build on earlier biochemical work in heterologous cell models , 15, 16 Earlier work had unequivocally shown that crosstalk between SOCE and ADCY8-dependent cAMP signaling was coordinated locally within cellular membranes and likely within membranes enriched in lipid rafts , 11, 12,2+/CaM-activated PDEs  including macrophages, cardiac, and neuronal cell types (2+) store depletion regulates their activities and cellular functions.Although our findings are most relevant to HASMCs, we predict that other cell types that express ADCY8 and PDE1C may avail themselves of the signaling dynamics reported here to efficiently regulate SOCE and coordinate localized effects of SOCE on ADCY8- and PKA-regulated functions. Indeed, certain nonexcitable cells, including macrophages and glioblastoma cells, also express Cated PDEs , 41. Simted PDEs . Based oer study . In addill types . Because2+-dependent activation of PDE1C as a “trigger” that promotes optimal SOCE-mediated control of ADCY8 in HASMCs is likely best viewed in the context of recent work from Zhang et al.  store depletion, in recruiting the PKA-inhibited fraction of Orai1 into SOCE or perhaps in prolonging Orai1 opening during SOCE. Of course, PDE1C could subserve both these roles in cells. Thus, we suggest that our finding represents a critical further step in this process, one that represents a Ca2+-dependent system through which cells could reduce PKA phosphorylation of Orai1 and consequently promote recruitment of Orai1 into a “new” round of SOCE or extend its activity. Thus, with analogy to the process described previously  and processed as described in . On threi.e., PDE activity) was determined as previously described , a gift from Dr James Guy Breitenbucher (Dart Neuroscience) or PF-04827726 (1 μM), Sigma. To inhibit PDE3 or PDE4 activities, cilostamide (5 μM), Cedarlane, or Ro, 20 to 1724 (10 μM), Calbiochem, were used, respectively.PDE-catalyzed cAMP hydrolysis , centrifuged at 10,000 RPM, and a fraction of the supernatant was collected for analysis of total lysate (input). To reduce nonspecific binding, lysates were precleared with protein A/G plus beads (40 μl bed volume: Santa Cruz) for 3 h with anti-IgG goat (1 μg/ml: Santa Cruz). Following centrifugation (5000 RPM), lysates were collected and immunoprecipitated with 1 μg/ml of anti- PDE1C (Santa Cruz) with protein A/G plus beads (40 μl bed volume) for 16 h at 4 °C. The beads were washed 3X with Triton lysis buffer, and protein was eluted at 37 °C for 30 min followed by immunoblotting. Antibodies and working concentrations for immunoblotting are indicated in the 2-diameter BD Falcon FluoroBlok cell culture inserts forming a monolayer (3 μm) to investigate leading edge protrusion as previously conducted (2-diameter (3 μm) BD Falcon cell culture inserts and imaged as indicated above.HASMCs resuspended in SMBM were plated on the upper surface of gelatin-coated [ddH2O supplemented with 0.25% gelatin (Biorad)], 24 mmonducted , 48. Che2-diameter BD Falcon cell culture inserts 72 h postinfection. Following plating, HASMCs on inserts in SMBM containing media, 0.5% FBS diluted in SMBM was added to the bottom of the transwell, and cells were allowed to extend leading edge protrusions for 4 h. Specific proteins were visualized by fixing the inserts with paraformaldehyde [4% (vol/vol)], rinsed with HBSS, and incubated for 1 h with the following primary antibodies: anti-c-myc mouse monoclonal (Sigma 1:1000), anti-GFP monoclonal rabbit (Santa Cruz 1:1000), anti-Flag M2 monoclonal mouse (Sigma 1:1000), for 1 h at room temperature or anti-AKAP79 rabbit polyclonal  at 4 °C for 16 h. The inserts were then washed with HBSS and incubated with fluorescently labeled Alexa-conjugated (488 nm) secondary antibodies and with phalloidin-tetramethylrhodamine B isothiocyanate (1:1000) and DAPI (1:1000) (0.3% BSA diluted in HBSS). The inserts were mounted on glass slide, and protein localization at the leading edge was visualized using a Leica TCS SP8 confocal laser scanning microscope.Protein localization in leading edge protrusions or cell bodies was visualized by transiently transfecting a monolayer of HASMCs with 2 μg of DNA plasmids   was added to the buffer. The cells were then washed with Krebs and kept at room temperature for an additional 30 min before imaging. Fluorescence emitted from Fura-2 AM was captured with an InCyt dual-wavelength imaging system (Intracellular Imaging) and a PixelFly CCD camera (1360 × 1024 resolution) mounted on a Nikon Eclipse TS100 (Nikon). HASMCs were perfused at a flowrate of 3 ml/min and were allowed to equilibrate for >5 min before data collection. Data were collected at 0.167 Hz. Cell viability was determined by a brief (15 s) application of 5 μM ionomycin (Sigma) at the end of the experiment. The change in the ratio induced during store depletion by 10 μM cyclopiazonic acid  in Ca2+ free Krebs buffer, and the change in the ratio induced by SOCE was determined as the difference between the peak during Ca2+ free Krebs + CPA and the trough before bath application of Ca2+ free Krebs + CPA and the difference between the peak during Krebs + CPA and the trough before bath application of Krebs + CPA, respectively. The rate in change during store depletion (Ca2+ free + CPA) and SOCE (Krebs + CPA) was determined as the difference in the ratio value between the peak drug response and the trough before drug response/the time difference between the point at which the drug has reached the peak response and the trough time point before drug response.Measurement of , rinsed with HBSS and incubated for 1 h at room temperature with the following primary antibodies: anti-c-myc mouse monoclonal (Sigma 1:1000) and anti-GFP monoclonal rabbit (Santa Cruz 1:1000). The coverslips were then washed with HBSS and incubated with fluorescently labeled Alexa-conjugated (488 nm) secondary antibodies and with phalloidin-tetramethylrhodamine B isothiocyanate (1:1000) and DAPI (1:1000) (0.3% BSA diluted in HBSS). Imaging was performed using the Leica TCS SP8 confocal microscope (Leica). White light laser (WLL) system was adjusted with settings for the laser line 499 nm  and 572 nm  and UV laser (405 nm) to identify DAPI nucleolus. Individual cells were imaged through a z-stack  acquisition mode using the HC PL APO CS2 63×/1.40 oil objective. The analysis was performed with the LAS-X Software (Leica Microsystems). To measure the relative mobilization distance of GFP-STIM1 to myc- Orai1 (cell surface), three straight ROIs were drawn per cell initiating it at the central point of each nucleolus and extending to the last bright point representing the cell surface. The intensity of each marker  was measured through the ROI (see HASMCs were transfected with myc-ORAI1 and GFP-STIM1 using 2 μg of DNA using the HASMC nucleofector kit (Lonza) according to the manufacturer’s instructions and the Nucleofector 2b Device (Lonza). Following 24 h post-DNA transfection, cells were plated on gelatin-coated coverslips (ddH ROI see . Any intt tests. When multiple groups were compared, statistical significance was analyzed using either a 1- or a 2- way analysis of variance, as appropriate, followed by an appropriate post hoc test as indicated in individual figure captions. In this manuscript, p ≤ 0.05 was considered significant.All data were analyzed statistically using GraphPad Prism Software. Throughout the manuscript, data are presented as means ± SEM. Statistical differences between two groups were obtained from unpaired, two-tailed Student’s All the data are contained within the manuscript and supplemental information.This article contains None declared.


Ataxia telangiectasia mutated (ATM) is a central kinase that activates an extensive network of responses to cellular stress via a signaling role. ATM is activated by DNA double strand breaks (DSBs) and by oxidative stress, subsequently phosphorylating a plethora of target proteins. In the last several decades, newly developed molecular biological techniques have uncovered multiple roles of ATM in response to DNA damage—e.g., DSB repair, cell cycle checkpoint arrest, apoptosis, and transcription arrest. Combinational dysfunction of these stress responses impairs the accuracy of repair, consequently leading to dramatic sensitivity to ionizing radiation (IR) in ataxia telangiectasia (A-T) cells. In this review, we summarize the roles of ATM that focus on DSB repair. Ataxia telangiectasia (A-T) was identified as a human disorder displaying radiosensitivity at both the cellular and clinical level in 1975 . The chaAs techniques to monitor DNA DSB repair improved and primary rather than immortalised cells were used, A-T was identified as having a subtle but significant defect in the repair of a subset of radiation induced DSBs in non-cycling cells . HoweverThe primary activation mode of ATM kinase activity is its dissociation from a dimeric form by autophosphorylation at Ser1981. In 2003, Kastan’s group elegantly demonstrated that the ATM dimer is autophosphorylated in response to DNA damage, subsequently the active form of an ATM monomer promotes the downstream phosphorylation and interaction with ATM partners . More reBiologically, several different activation modes have been reported. A DSB is the primary form of triggering ATM activation by dimer dissociation. The MRE11/RAD50/NBS1 (MRN) complex acts as the DSB sensor, thereby recruiting ATM at DSB sites, particularly by an interaction between ATM and Nijmegen breakage syndrome protein 1 (NBS1), resulting in further accumulation of ATM signaling and positive feedback ,13,14 F. ATM becIn addition, ATM can be activated under conditions of oxidative stress following the formation of dimers via disulfide-crosslinks . This moATM lies at the heart of a signal transduction process alerting cells to the presence of DSBs. The most significant aspect of this is to alter the chromatin in the DSB vicinity. For this process, multiple repair mediators—mediator of DNA damage checkpoint protein 1 (MDC1), RING finger ubiquitin E3 ligase (RNF8), RING finger 168 (RNF168), breast cancer susceptibility protein 1 (BRCA1), and p53 binding protein 1 (53BP1)—are recruited in an ATM-dependent manner . An important role of ATM is the activation of cell cycle checkpoint arrest . In some cell types, apoptosis can also be activated if an excessive amount of DNA damage is induced. Central to checkpoint activation is the ATM-dependent phosphorylation of Chk2 (see Bartek & Lukas for a review) . AlthougIn parallel to cell cycle arrest and DSB repair, the transcription machinery is also arrested to prevent aberrant mRNA synthesis at damaged transcription regulatory sequences. ATM has a critical role in preventing transcription after DSB formation by RNA polymerase II (RNAPII) that has responsibility for mRNA synthesis at gene loci, and RNA polymerase I that synthesizes ribosomal RNA ,37. ATM-Although initial studies suggested that the level of DSB repair was normal in A-T deficient cells, accumulating evidence clearly shows that lack of ATM kinase activity causes a defined DSB repair defect . NonetheATM plays a role in DSB end resection, which affects the fidelity of DSB repair. The dysregulation of resection impairs the progression of homologous recombination (HR) in S/G2 phase. The rejoining by these inefficiently resected DSBs by NHEJ likely leads to deletion mutations. In S/G2 phase, the signaling machinery switches from ATM to ATR in concert with the progression of resection because ATM is primarily activated at unresected DSB ends. In contrast, ATR is activated on ssDNA-replication protein A (RPA) following resection ,49. TherIn response to DSB induction in S/G2 phase, ATM is rapidly activated, similar to the situation in G1 phase inducing the recruitment of DDR responders and promoting foci formation such as γH2AX, MDC1, and 53BP1, followed by downstream signaling. 53BP1 hyper-phosphorylation, strictly dependent on ATM, occurs in S/G2 and G1 phases . The hypIn parallel to the progression of resection, ATM also controls CtIP activity to restrict excessive resection. A recent study showed that protein inhibitor of activated STAT4 (PIAS4) dependent SUMOylation of CtIP promotes RNF4 dependent CtIP ubiquitination, which leads to its degradation  Figure . ATM-depThe regulation of DNA-replication-fork-associated single-ended DSBs (seDSBs) in S phase is different from two-ended DSBs because seDSBs must be directed toward HR because the lack of a DSB counterpart at seDSBs leads to chromosomal translocation if NHEJ is used. Consistent with this notion, lack of ATM activity leads to toxic LIG4-mediated chromosome fusions after DNA-replication-fork-associated seDSBs . ApproprTo summarise this section, ATM has a critical role in regulating resection at DSBs, which arises via complex regulation of multiple substrates and critically depends on the regulation of chromatin structure at the DSB site. Not surprisingly, this impact of ATM affects DSBs in diverse ways. One affect is a failure to activate HR in G2 phase. However, although less dramatic, resection also arises in G1 phase, impacting upon repair via NHEJ. Here, two forms of NHEJ have been described, resection independent and dependent, and the latter is ATM-dependent in G1 phase . FailureThe details above reveal the significant functions that ATM has in determining the response to DSBs and its influence on how they are repaired. Here, we describe how ATM is essential for the repair of a small fraction of DSBs, its requirement for cell cycle checkpoint arrest, transcriptional repression and resection, and hence an influence on pathway choice, at DSBs . As brie


CaCCs are the translated products of two members  of the Anoctamin family of genes comprising ten paralogs. This review focuses on recent progress in understanding the molecular mechanisms involved in the regulation of ANO1 by cytoplasmic Ca2+, post-translational modifications, and how the channel protein interacts with membrane lipids and protein partners. After first reviewing the basic properties of native CaCCs, we then present a brief historical perspective highlighting controversies about their molecular identity in native cells. This is followed by a summary of the fundamental biophysical and structural properties of ANO1. We specifically address whether the channel is directly activated by internal Ca2+ or indirectly through the intervention of the Ca2+-binding protein Calmodulin (CaM), and the structural domains responsible for Ca2+- and voltage-dependent gating. We then review the regulation of ANO1 by internal ATP, Calmodulin-dependent protein kinase II-(CaMKII)-mediated phosphorylation and phosphatase activity, membrane lipids such as the phospholipid phosphatidyl--bisphosphate (PIP2), free fatty acids and cholesterol, and the cytoskeleton. The article ends with a survey of physical and functional interactions of ANO1 with other membrane proteins such as CLCA1/2, inositol trisphosphate and ryanodine receptors in the endoplasmic reticulum, several members of the TRP channel family, and the ancillary Κ+ channel β subunits KCNE1/5.Ca Slow kinetics, outward rectification and a reversal potential near the equilibrium potential for Cl− (ECl) are hallmark properties of the native CaCCs of interest in this review.A chloride conductance activated by a physiological rise in intracellular Ca oocytes . Ιn oocyechanism . Ca2+-ac neurons , cardiac neurons , skeleta neurons  and smoo neurons  cells, e neurons ,26, vasc neurons ,28, exoc neurons , various neurons , mast ce neurons , hepatoc neurons , and man~ 150 nM . At phys2+ concentration in the perfusate above ~ 150 nM. Single-channel experiments in these cell types revealed that CaCCs are small conductance channels (~ 3 pS) and, like macroscopic ICl(Ca) [2+ concentration on the inner side of the membrane ([Ca2+]i), so that the I–V relationship becomes almost linear at concentrations above 1 μM. The Kd for Ca2+-mediated channel opening decreases with membrane depolarization i < ~ 1 μM, and are time- and voltage-sensitive. Another argument against Bestrophins encoding classical CaCCs was the observation that in rat mesenteric arterial smooth muscle cells, the Ca2+- and cGMP-sensitive but voltage-insensitive Bestrophin 3 current coexists with the classical time- and voltage-dependent CaCC current i < 1 μM (termed “Ca2+-dependent sensitization of activation” or CDSA) and decreasing channel activity at [Ca2+]i > 10 μM through CaM-dependent inactivation or CDI. Deletion of splice segment a resulted in loss of binding of apoCaM and disappearance of both CDSA and CDI, while the exclusion of segment b selectively suppressed CDI. In conclusion, tethered or freely diffusing CaM is not required to activate the channels, but it is possible that CaM may act as a modulator of ANO1.Yang and Colecraft  concludeal study , that CadK for Ca2+ and Hill coefficient (> 2) of CaCCs in Xenopus oocytes i). They showed that blocking CaMKII with KN-93 or Autocamtide-2-related inhibitory peptide (ARIP) increased the magnitude of ICl(Ca) in coronary and pulmonary artery myocytes dialyzed with [Ca2+]i in the 500–1000 nM Ca2+ range. This effect was consistent with a modulation of the gating properties of the channels as evidenced by the noted acceleration of the slow current relaxation during depolarizing steps, slower deactivation during repolarizing steps, and a leftward shift in the steady-state activation curve. Cell dialysis with a constitutively active Ca2+-independent CaMKII isoform (AutoCaMKII) produced opposite effects.Greenwood et al.  examinedCl(Ca) in a small fraction of cells. Thus, the authors could only speculate that the differential effects of CaMKII-mediated phosphorylation of CaCCs in arterial and venous myocytes might be attributed to a differential pattern of CaMKII isoforms expression (and likely phosphatases) and/or CaMKII phosphorylating unidentified regulators of CaCCs in the two cell types.Interestingly, in portal vein myocytes, such a mode of regulation by CaMKII was not detected, and CaMKII appeared to instead enhance Iet al. i (2 modestly inhibited ANO2 (or TMEM16B), and this effect was Ca2+-independent. Co-expression of ANO1 and Danio rerio voltage-sensitive phosphatase (Dr-VSP), which depletes endogenous PIP2 when the membrane is depolarized i . In contolarized , produceet al. i (100 μM).A subsequent study by De Jesús Pérez et al.  showed t2 depletion or a change in the membrane distribution of ANO1 due to altered trafficking.These investigators also examined whether fatty acids (FA), phosphatidylserine (PS), and cholesterol (discussed in the next section) regulate ANO1. They found that stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids, and methyl stearate and PS all inhibit ANO1 in a concentration- and voltage-dependent manner. These effects were attributed to a direct membrane-delimited lipid-channel protein interaction and were not associated with PIPet al. i. This arrangement would help minimize the potentially negative impact of self-sustaining positive feedback loops between ANO1 and VGCCs that could lead to neuronal hypersensitivity i. Subsequent studies showed that ANO1 is responsible for ICl(Ca) in airway myocytes and is involved in smooth muscle contraction to agonists and airway hypercontractility in asthma i (≤ ~ 1 μΜ) when intracellular ATP, which is the main phosphate donor in the biosynthesis of PIP2, is removed (2, inhibiting or supporting ANO1 activity depending on certain conditions, or could the regulation of KCNE proteins by PIP2 be responsible for these differences? Does alternative splicing alter the regulation of ANO1 by PIP2, and what is the reciprocal influence of CaMKII-induced phosphorylation and regulation by PIP2 of ANO1 in vivo?Another critical unanswered question concerns the regulation of ANO1 by PIP removed  [57–59,1− in different compartments of the cell and organelles also playing a key role? Finally, is the regulation of ANO1 by the various signal transduction pathways discussed in this article altered and contributing to disease states? The next decade will undoubtedly be exciting as it will raise the fog on many of these questions and bring new challenging puzzles to resolve regarding the biological roles of ANO1 and its paralogs in health and disease.CaCCs encoded by ANO1 have been implicated in many diseases such as systemic  and pulm


However, blockade of voltage-dependent Ca2+ channels (VDCCs) did not uncover direct preNMDAR-mediated Ca2+ transients in the axon. This suggests that astrocyte-mediated glutamate release onto preNMDARs does not result in a direct Ca2+ influx, but that it instead leads to an indirect interaction with presynaptic VDCCs, boosting axonal Ca2+ influx. These results reveal one of the main remaining missing pieces in the signaling cascade of t-LTD at developing cortical synapses.Spike-timing-dependent long-term depression (t-LTD) of glutamatergic layer (L)4-L2/3 synapses in developing neocortex requires activation of astrocytes by endocannabinoids (eCBs), which release glutamate onto presynaptic NMDA receptors (preNMDARs). The exact function of preNMDARs in this context is still elusive and strongly debated. To elucidate their function, we show that bath application of the eCB 2-arachidonylglycerol (2-AG) induces a preNMDAR-dependent form of chemically induced LTD (eCB-LTD) in L2/3 pyramidal neurons in the juvenile somatosensory cortex of rats. Presynaptic Ca Presynaptic NMDA receptors (preNMDARs) have important functions in synaptic transmission, information processing, and long-term plasticity in several regions of the brain –3. PartiUnder certain conditions, preNMDARs in barrel cortex can also function as autoreceptors for presynaptically released glutamate. Bursts of APs followed by a correctly timed additional AP can induce pattern-dependent LTD at the L4-L2/3 synapse . Importa2+ signals mediated by preNMDARs. Lack of axonal Ca2+ signals in boutons of L5 and L4 neurons in developing neocortex upon iontophoresis of aspartate or upon MNI-glutamate uncaging, and lack of an APV sensitive component in single AP-evoked Ca2+ transients argue against the existence of preNMDARs cyclohepten-5,10-imine maleate (MK-801), and L-glutamic acid from Tocris, FK506 from Abmole Bioscience.Chemicals were obtained from the following sources: 2-AG and cyclosporin-A from Sigma-Aldrich, 1--5-(4-iodophenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (AM251), d-AP5 from Ascent Scientific, (+)-5-methyl-10,11-dihydro-2+ imaging from presynaptic boutons during t-LTD induction would be the best approach. However, this is technically challenging, since it requires imaging from the presynaptic bouton of an identified synaptic connection while at the same time controlling AP firing in the pre- and postsynaptic neuron. As an alternative approach, we tested if direct bath-application of 2-AG resulted in synaptic depression without postsynaptic activity at L4-L2/3 synapses, similar to what was previously described for L5-L5 synapses [μM) for 20 min while continuing presynaptic stimulation at 0.1 Hz. We observed a long-lasting depression of the EPSPs 10–30 min after the washout of 2-AG  that decayed back to baseline levels after wash-out , while infusing a control intracellular solution into the astrocytes resulted in eCB-LTD , while infusion of MK801 into the postsynaptic cell had no effect on eCB-LTD  and cyclosporin-A (25 μM) at least for 1 h before the start of the experiment and with 20 μM and 10 μM, respectively, during the experiment. EPSPs were evoked by extracellular stimulation in L4, and 2-AG was washed-in for 20 min as described above. In the condition of blocked calcineurin activity, no eCB-LTD was induced  and the morphological dye Alexa-594 (50 μM) to trace the axon to L2/3  in the presence of 2-AG. This number was not different from spontaneous events before 2-AG application (8 events in 40 min). However, comparing the time course of the presynaptic AP-evoked Ca2+ transients before and after 2-AG application revealed a prolongation of the AP-evoked Ca2+ signal in the presence of 2-AG . Furthermore, single AP-evoked presynaptic Ca2+ signals alone were not influenced by blocking NMDA receptors. Bath-application of APV did not change the peak amplitude of the Ca2+ transients , nor its decay  in spiny stellate axons before and during bath application of 2-AG , while having no effect on the rest  abolished the effect of bath-application of 2-AG on the AP-evoked Ca2+ transients , excluding a nonspecific effect of 2-AG. Furthermore, repeating the experiment without any drug application had no effect on the AP-evoked Ca2+ transients  demonstrating long-term stability of the experimental design and ruling out any time-dependent changes in axonal Ca2+ buffering due to the Ca2+ indicator.Therefore, in order to investigate the phenomenon of the specific 2-AG induced broadening of the AP-evoked Ca Figures . We normansients . In cont+ signal . Fitting2+ signaling in cerebellar stellate cells [2+ dynamics. Thus, our experiments revealed an APV-sensitive presynaptic Ca2+ component that manifested itself in a broadening of AP-evoked Ca2+ transients.2-AG evoked release of glutamate might activate dendritic NMDARs on the recorded neuron, which could potentially influence somatic membrane potential, AP generation, and somatic AP properties. Previously, such somato-dendritic NMDAR activation was shown to influence presynaptic Cate cells . However2+ signal. A burst of APs at 100 Hz followed by a single AP 50 ms later evoked an axonal Ca2+ transient that was more rapidly decaying after wash-in of APV . Thus, the additional AP that followed 50 ms after the burst of 3 APs leads to a significant (p < 0.05 by one-way ANOVA) additional presynaptic Ca2+ influx that was abolished by APV. The requirement of the presence of this delayed 4th AP suggests an interaction of the preNMDARs with a voltage-dependent mechanism initiated by the additional AP.Recently, it was shown that specific patterns of presynaptic activity alone, consisting of a burst of APs at 100 Hz or above, followed by a single AP between 50 and 200 ms later, can induce LTD. This form of LTD requires the activation of preNMDARs, but does not depend on astrocyte activation [n of APV . The dif2+ transients, which results in an additional axonal Ca2+ influx that is intrinsically linked to the presence of the presynaptic AP. The source of the glutamate that activates the preNMDARs can either originate from 2-AG activated astrocytes or from glutamate spillover by a specifically timed presynaptic burst of APs.In summary, our experiments suggest that the activation of preNMDARs causes a slowing of the decay of AP-evoked Ca2+ transient could directly be due to the preNMDARs, which could contribute to Ca2+ influx during the AP-evoked axonal membrane depolarization and the subsequent relief of the Mg2+ block. In this scenario, preNMDARs would function as classical coincidence detectors, similar to what has been suggested for postsynaptic NMDARs. However, it has been shown that preNMDARs during early development might contain the GluN3A subunit, which renders them largely insensitive to Mg2+ block and with low permeability for Ca2+ [+ influx or to exert a metabotropic effect during their activation. Both mechanisms could activate voltage-dependent Ca2+ channels (VDCCs) beyond the membrane depolarization caused by the axonal AP and contribute to an additional Ca2+ influx. To distinguish between a direct Ca2+ influx through preNMDARs and an interaction with VDCCs, we performed axonal Ca2+ imaging as described above and blocked VDCCs (2+ transient which was abolished in the presence of the VDCC-blockers Cd2+ (100 μM) and Ni+ (50 μM) even though the somatic AP waveform was unchanged . Subsequent continuation of somatic AP stimulation during bath application of 2-AG, but still in the presence of Cd2+/Ni+ did not uncover an AP-evoked Ca2+ transient through preNMDARs . This observation suggests that axonal membrane depolarization is not required for unblocking preNMDARs from a putative Mg2+ block to render them permeable for Ca2+. Contrary, we conclude that functional VDCCs are required so that preNMDARs can interact with them to prolong the axonal Ca2+ influx.The broadening of the AP-evoked presynaptic Cafor Ca2+ . An alteed VDCCs . Somatic2+ signaling and the activation of preNMDARs, thereby showing an overlapping mechanism of induction with t-LTD [2+ dynamics during the induction of eCB-LTD allowed us to investigate the functional consequences of preNMDAR activation. In line with other studies, we found no evidence for a direct Ca2+ influx through preNMDARs during eCB-LTD induction or glutamate iontophoresis [2+ influx through these channels. Thus, we conclude that the action of preNMDARs has an indirect influence on presynaptic Ca2+ transients by interacting with VDCCs. These findings can reconcile some of the controversial results regarding preNMDARs and are consistent with the electrophysiological evidence for their influence in t-LTD.NMDARs are essential ionotropic glutamate receptors for synaptic transmission, information processing, and synaptic plasticity. While classically thought to be located mainly postsynaptically, there is growing anatomical and physiological evidence that NMDARs also have important functions at presynaptic sites , 31–38. phoresis , 14. Ins2+ through VDCCs, which is thought to prime phospholipase C (PLC), which is subsequently activated by the presynaptic release of glutamate binding to the metabotropic glutamate receptor type 5 (mGluR5) [2+ activity is necessary. Furthermore, preNMDARs are required for both t-LTD and eCB-LTD. preNMDARs are expressed in a target-cell-specific way only at a subset of synapses. This suggests that preNMDAR-mediated plasticity is limited to specific neuronal connections [2+ transients in a subset of the investigated axonal boutons.The chemically induced eCB-LTD presented here shares the same signaling cascade as found in t-LTD. In t-LTD, the eCB 2-AG is synthetized by postsynaptic AP firing followed by presynaptic glutamate release. The postsynaptic backpropagating AP evokes an increase in postsynaptic Ca(mGluR5) , 7. In enections , 39–41. 2+ signal. This is in line with our earlier observation that when LTD is induced by direct electrical stimulation of astrocytes , this LTD still requires presynaptic AP firing during the astrocyte activation [2+ dynamics. This in turn presumably leads to calcineurin modulation and LTD. Interestingly, very similar results have been obtained by others. In the first study showing involvement of eCBs and preNMDARs in t-LTD, it was already shown that eCB application only led to LTD if it was paired with presynaptic activity [The presynaptic AP is an essential component for eCB-LTD induction, since without the interaction of the AP with preNMDAR activation, there is no change in the presynaptic Cativation . This obactivity . Furtheractivity .2+ dynamics at presynaptic sites. Furthermore, several studies have shown that somatic depolarization can lead to an additional axonal depolarization that gives rise to graded, analog release of transmitter [2+ signals in our experiments.A similar interaction of a presynaptic ionotropic glutamate receptor being activated by astrocytes and influencing synaptic release has recently been demonstrated . In thisnsmitter , 44. Imp2+ dynamics has also been described [2+ transient and increase neurotransmitter release. However, we deem it unlikely that a similar mechanism involving postsynaptic NMDAR activation can explain our observations. First, experiments with MK801 in the pre- or postsynaptic neuron show that both t-LTD [2+ transient broadening mediated by postsynaptic NMDARs was only observed with repetitive AP firing in the absence of extracellular Mg2+ [2+ transients in L4 boutons occurred with single AP firing in the presence of 1 mM extracellular Mg2+. Under our experimental conditions, the potassium efflux through postsynaptic NMDARs is likely minimal due to Mg2+ block of these receptors. Finally, we observed that not all L4 boutons were showing a 2-AG induced broadening of the presynaptic Ca2+ transient. This is similar to what was observed in excitatory boutons in L5 of developing neocortex [2+ transient broadening such a lack of effect in some boutons is harder to explain, since postsynaptic NMDARs are ubiquitously expressed at most glutamatergic synapses [It should be noted that an intriguing interaction of postsynaptic NMDAR activation with presynaptic Caescribed . At hippth t-LTD , 8 and eth t-LTD . Therefolar Mg2+ . In conteocortex . It indisynapses –49.2+ imaging, we were able to show that the p-LTD presynaptic activity pattern evoked an APV-sensitive Ca2+ component, whereas a burst of 3 APs alone did not. Thus, preNMDARs can differentially be activated depending on the pattern of presynaptic activity and only contribute to an additional Ca2+ influx under certain conditions.It was recently shown that a presynaptic burst of APs followed by a single AP between 50–200 ms later can also trigger LTD  . The preAn interesting question in this context is which type of VDCC is modulated by the preNMDARs? Previous experiments suggest that neither L-type nor R-and T-type VDCCs are required, because t-LTD can be induced in the presence of blockers of these channels using a burst of 3 postsynaptic APs followed by a single presynaptic AP at -10 ms . Single + influx through the receptor plays a role in the interaction of preNMDARs with VDCCs. A similar conclusion on the importance of NMDAR-mediated Na+ influx was reached for the effect of preNMDARs on spontaneous synaptic release [Our data is in line with the idea that preNMDAR-mediated depolarization of the terminal carried by axonal Na release . This io release , 50. Sim release . This ef release , 51 unli2+ block by a backpropagating AP to supralinearly enhance postsynaptic Ca2+ influx [2+ signal. This is in line with several findings about the subunit composition of preNMDARs at developing synapses in the neocortex. In the developing visual cortex, preNMDARs contain the NR3A subunit rendering these NMDARs insensitive to Mg2+ and little Ca2+ permeable [2+ influx through preNMDARs. The lack of a presynaptic Ca2+ signal by iontophoresing glutamate onto presynaptic boutons or by uncaging of MNI-glutamate was interpreted by others as a lack of preNMDARs [2+ signaling is rather subtle and becomes only apparent in the presence of specific patterns of presynaptic APs, thereby explaining the apparent lack of preNMDAR activity in other studies. Similar results were obtained by others when performing Ca2+ imaging experiments at glutamatergic synapses onto cortical interneurons: only prolonged axonal activation with sustained bursts of APs clearly uncovered an APV-sensitive component in the Ca2+ transient [2+ influx has been observed through preNMDARs suggesting that there is a synapse-specific differential subunit composition of preNMDARs [Several studies to date have sought for functional evidence of preNMDARs in neocortex and have come to the conclusion that these receptors do not exist , 14. Our+ influx . In contermeable , 40. BotreNMDARs , 14. Howransient . At othereNMDARs .2+ imaging data but also on the absence of L4-L2/3 t-LTD in a transgenic mouse in which L2/3 NMDARs were selectively disrupted [Importantly, the recent conclusion that t-LTD requires post- rather than presynaptic NMDARs was not just based on negative Caisrupted . Althougisrupted , thereby2+ influx or by an inactivation of presynaptic K+ channels, both of which could broaden the AP locally and thus enhance presynaptic Ca2+ influx [Finally, pharmacological evidence presented recently by Carter and Jahr  suggests+ influx , which w2+ influx. This modulation could be either by contributing to the local membrane depolarization or by a metabotropic action affecting VDCCs or other presynaptic ionic conductances. Either mechanism would affect subsequent axonal APs by modifying AP-induced Ca2+ dynamics, thereby leading to the induction of LTD. The elucidation of the mode of action of preNMDARs is one of the remaining missing pieces to understand the signaling cascade of t-LTD at developing cortical synapses.In summary, we show evidence for the existence of functional preNMDARs in spiny stellate axons at L4-L2/3 synapses in the developing rat barrel cortex. Their function is to sense glutamate either released from astrocytes or from spill-over by enhanced presynaptic activity and then to modulate the local axonal Ca


Cyniclomyces guttulatus is usually recognised as an inhabitant of the gastrointestinal (GI) tract in rabbits. However, large numbers of C. guttulatus are often detected in the faeces of diarrhoeic rabbits. The relationship of C. guttulatus with rabbit diarrhoea needs to be clearly identified. In this study, a C. guttulatus Zhejiang strain was isolated from a New Zealand White rabbit with severe diarrhoea and then inoculated into SPF New Zealand white rabbits alone or co-inoculated with Eimeriaintestinalis, another kind of pathogen in rabbits. Our results showed that the optimal culture medium pH and temperature for this yeast were pH 4.5 and 40–42 °C, respectively. The sequence lengths of the 18S and 26S ribosomal DNA fragments were 1559 bp and 632 bp, respectively, and showed 99.8% homology with the 18S ribosomal sequence of the NRRL Y-17561 isolate from dogs and 100% homology with the 26S ribosomal sequence of DPA-CGR1 and CGDPA-GP1 isolates from rabbits and guinea pigs, respectively. In animal experiments, the C. guttulatus Zhejiang strain was not pathogenic to healthy rabbits, even when 1 × 108 vegetative cells were used per rabbit. Surprisingly, rabbits inoculated with yeast showed a slightly better body weight gain and higher food intake. However, SPF rabbits co-inoculated with C. guttulatus and E. intestinalis developed more severe coccidiosis than rabbits inoculated with C. guttulatus or E. intestinalis alone. In addition, we surveyed the prevalence of C. guttulatus in rabbits and found that the positive rate was 83% in Zhejiang Province. In summary, the results indicated that C. guttulatus alone is not pathogenic to healthy rabbits, although might be an opportunistic pathogen when the digestive tract is damaged by other pathogens, such as coccidia. Lapine rotavirus), bacteria  and parasites  have been identified as pathogens which cause diarrhoea in rabbits [Cyniclomyces guttulatus, a commensal yeast in the rabbit gastrointestinal tract, is also commonly seen in diarrhoea cases. However, it is unclear whether this organism causes or is a co-cause of diarrhoea with other pathogens. Some researchers believe that C. guttulatus is not a pathogen which causes diarrhoea, but is probably a salubrious normal inhabitant based on its common existence in healthy animals and the absence of clinical signs in experimental rabbits inoculated with C. guttulatus isolates [C. guttulatus and diarrhoea in rabbits, a C. guttulatus strain was isolated and identified from a New Zealand White rabbit with severe diarrhoea, and the culture conditions were optimised. Then, its relationship with rabbit diarrhoea was investigated through the inoculation of C. guttulatus alone and coinoculation with an intestinal protozoan, Eimeria intestinalis. In addition, the prevalence of C. guttulatus in rabbits was surveyed in Zhejiang Province, China.Diarrhoea is very common in rabbits, especially in weanling rabbits, and causes huge losses in rabbit production. According to some reports, more than 50% of mortalities in rabbits may be caused by intestinal disease with diarrhoea ,2,3. Cur rabbits ,13,14,15isolates ,17. Howeisolates ,20,21,22C. guttulatus Zhejiang strain was isolated from a diarrhoeic rabbit, cultivated in YPG medium at low pH, and then identified by light microscopy. Microscopically, the vegetative cells of C. guttulatus were ellipsoid, colourless and approximately 20–50 μm in length and occupied two large vacuoles in the cytoplasm [C. guttulatus formed pseudohyphae when it was cultivated in a stationary culture station [C. guttulatus reached 4.62 × 107 ± 3.98 × 106 cells/mL over 60 h from the initial culture density of 1 × 104 cells/mL [A ytoplasm A,C. C. g station B. When i station D,E. The  station D. The lo station E. At a ccells/mL E.C. guttulatus Zhejiang strain was 1559 bp. It showed 98% sequence identity and 100% coverage with that of the C. guttulatus NRRL Y-17561 strain reported in GenBank (accession number JQ698886.1) [C. guttulatus CGDPA-GP1 and DPA-CGR1 strains originate from herbivores, guinea pigs and rabbits, respectively, whereas the C. guttulatus Dog-1 and DPA-CGD1 strains originate from dogs. Thus, the C. guttulatus Zhejiang strain showed a closer phylogenetic relationship with the strains originating from herbivores than those from carnivores.The sequence length of the 18S fragment of the 98886.1) A. In the98886.1) B. AccordC. guttulatus before the experiment. Two days after inoculation, C. guttulatus was found in rabbit faeces of all three groups (G1–G3) that were inoculated with 1 × 106, 1 × 107 and 108C. guttulatus vegetative cells per rabbit. However, no rabbits in these groups showed clinical signs of illness. Interestingly, the C. guttulatus-inoculated groups (G1–G3) had less feed waste than the control group (G4) [p > 0.05) [C. guttulatus cells in the gastric and intestinal contents. In particular, a thick layer of C. guttulatus cells colonised the gastric mucosa [C. guttulatus [C. guttulatus cells probably attached to the stomach mucosa through these filamentous saccharides, as shown by transmission electron microscopy [C. guttulatus Zhejiang strain colonised the gastric mucosa, but it was nonpathogenic to healthy rabbits.All SPF rabbits were healthy and were negative for oup (G4) B,C. The  > 0.05) A. Autopsc mucosa D–F. PAS-ttulatus G. The C.croscopy H,I. Our C. guttulatus and E. intestinalis before the experiment. Rabbits preinoculated with C. guttulatus (CG/EI group) developed more severe illness and intestinal lesions following E. intestinalis infection than those not inoculated with C. guttulatus (EI group). Compared with the EI group, more severe diarrhoea, loss of appetite and constipation were observed in the CG/EI group. The number of C. guttulatus vegetative cells in the faeces of the CG/EI group was significantly higher than that of the CG group (p < 0.05) at 9 and 10 days after E. intestinalis infection. No C. guttulatus was detected in the EI and NON groups [5 on day 10, 4.7-fold and 3.2-fold higher than those of the EI group, respectively. In contrast, coccidian reproduction was lower in co-inoculated rabbits than in rabbits inoculated with E. intestinalis alone, as shown by a markedly lower oocyst output in the CG/EI group than in the EI group; the total faecal oocyst count per rabbit on day 10 was 2.87 × 109 ± 8.13 × 107 in the coinoculated group compared with 4.57 × 109 ± 6.83 × 107 in the group without yeast inoculation. No E.intestinalis was detected in either the CG or NON groups [9 ± 2.45 × 107 on day 11 and 1.61 × 109 ± 3.43 × 107 on day 13). Faecal oocyst excretion in the CG/EI group peaked and cleared earlier than that in the EI group [C. guttulatus proliferation was enhanced by E. intestinalis infection, whereas E. intestinalis reproduction was suppressed by C. guttulatus. In addition, one rabbit in the CG/EI group died on the 10th day post-infection. The dead rabbit had disseminated haemorrhage and nodules in the lower jejunum and ileum, and a large number of E. intestinalis oocysts and C. guttulatus vegetative cells were detected in the small intestine [E. intestinalis and C. guttulatus developed more severe clinical signs and intestinal lesions (and one death) than rabbits inoculated with E. intestinalis only, associated with higher C. guttulatus and lower E. intestinalis output. The findings suggested that C. guttulatus might be an opportunistic pathogen in rabbits with coccidia.All SPF rabbits were healthy and were negative for N groups A. The meN groups B. In addEI group B. Thus, ntestine D,E. In antestine C. In sumC. guttulatus by analysing C. guttulatus cells in faeces. C. guttulatus was detected in 210 of 253 faecal samples from rabbits over 30 days old in Zhejiang Province, and the positive rate was 83%. In this survey, the positive rate for rabbits over 60 days old was 69.7% (46/66), and for rabbits under than 60 days old, it was 87.7% (164/187). The number of rabbits with a high C. guttulatus load (>100 cells per microscopic field at 200× magnification) was 59 (23.3%), consisting of 4 rabbits under 60 days old and 55 rabbits over 60 days old . Thus, C. guttulatus was highly prevalent in healthy rabbits, especially in older rabbits.We conducted a survey of rabbits carrying C. guttulatus is a monotypic yeast genus of the Saccharomycetaceae family and inhabits the GI tract of many animal species, including rabbits, dogs and guinea pigs [C. guttulatus was first described more than 60 years ago and is commonly found in rabbit faeces. A large number of C. guttulatus cells are often found in the faeces of diarrhoeic rabbits, but it is unknown whether C. guttulatus causes diarrhoea in rabbits. We isolated a C. guttulatus Zhejiang strain from a rabbit with severe diarrhoea. At optimised culture pH and temperature, a single clone was expanded and studied for its pathogenicity in rabbits. We demonstrated that the C. guttulatus Zhejiang strain is not a primary pathogen which causes diarrhoea in healthy SPF rabbits. An inoculation rate as high as 1×108 vegetative cells per rabbit did not result in any clinical signs of illness or gastrointestinal lesions. This is consistent with previously reported studies, where rabbits orally or intravenously inoculated with C. guttulatus isolates showed no clinical signs of illness [C. guttulatus showed better performance in terms of body weight gain and food intake. C. guttulatus seems to be a probiotic microorganism in rabbits, especially in weanling rabbits. However, the abundant vegetative cells of C. guttulatus commonly seen in the faeces of rabbits with diarrhoea suggest that it may be a causative microorganism of GI tract disturbance.nea pigs ,22,23. C illness ,17,24. UC. guttulatus could be an opportunistic pathogen or play a co-causative role in diarrhoea of its host based on indirect evidence that anti-fungal agents, such as nystatin, are effective in the treatment of some diarrhoeic cases [C. guttulatus was proven to be an opportunist through coinfection with the coccidian species E. intestinalis, a parasite which causes diarrhoea and intestinal lesions in rabbits. Mortality and more severe clinical signs and intestinal lesions were observed in rabbits coinfected with C. guttulatus and E. intestinalis than in rabbits inoculated with E. intestinalis only. Compared with rabbits inoculated with C. guttulatus alone, vegetative cells of C. guttulatus prolifically multiplied in the coinfection group, peaking at 9–10 days post-E. intestinalis infection. This time period is consistent with that of intestinal lesions of E. intestinalis infection [C. guttulatus was probably a consequence of the altered GI environment in the rabbits from E. intestinalis infection. Compared with rabbits with E. intestinalis infection alone, E. intestinalis oocyst excretion in coinfected rabbits decreased by 37%. We speculate that the rapid and considerable multiplication of C. guttulatus vegetative cells could contribute to severe symptoms in coinfected rabbits, and this multiplication of C. guttulatus also limited the reproduction of E. intestinalis.Some authors have proposed that ic cases ,20,21,22nfection ,26. The C. guttulatus was prevalent in rabbits in Zhejiang, China. The positive rate in rabbits was as high as 83%. This rate was significantly higher than that in other host animals, such as dogs, in which a prevalence of 14–21% has been reported [C. guttulatus may contribute to the morbidity and mortality of rabbits with coccidiosis.In addition, our epidemiological survey showed that reported ,21,27. Creported . TherefoC. guttulatus and rabbit diarrhoea was investigated. A C. guttulatus strain was first isolated and identified from a diarrhoeic rabbit with special YPG medium (pH 1.5). To investigate the pathogenicity of C. guttulatus alone on rabbits, sixteen SPF rabbits were randomly divided into 4 groups with 4 rabbits per group. Three groups of SPF rabbits were inoculated with 1 × 106, 1 × 107 or 1 × 108C. guttulatus vegetative cells per rabbit. One group was used as an uninoculated control. The physical state of the rabbits and possible clinical signs were observed every day post-inoculation. To observe possible pathological changes in the GI tract, all of the experimental rabbits were administered dexmedetomidine hydrochloride  and tiletamine hydrochloride-zolamide hydrochloride  before euthanasia through air injection  with sterile syringes  in the animal diagnosis and treatment centre of our institute 18 days after inoculation according to the method of Close B. (1997) [C. guttulatus is an opportunistic pathogen, another sixteen SPF rabbits were used. They were divided into 4 groups with 4 rabbits per group. Two groups of SPF rabbits were inoculated with 1 × 107C. guttulatus vegetative cells per rabbit, and the other two groups were uninoculated. Fourteen days later, one of the inoculated and uninoculated groups was infected with 1×104E. intestinalis oocysts per rabbit. Clinical signs, oocyst output and C. guttulatus cells in faeces were examined post-inoculation/infection. In the second part, to survey the prevalence of C. guttulatus in rabbits, fresh faecal samples collected from rabbitries in Zhejiang Province, China were examined.This study was divided into two parts. In the first part, the relationship between . (1997) . Then, tC. guttulatus during transportation, and the faeces of the experimental rabbits were examined every day under microscopy to check the existence of oocysts. They were reared within RBC6H5-independent ventilation cages  were acquired from Pizhou Dongfang Rabbit Breeding Co., Ltd.  and reared in our institute. Three days before the experiment, all rabbits were given itraconazole with free access to water by adding a dosage of 100 mg/L to avoid contamination by C. guttulatus Zhejiang strain was isolated from a severely diarrhoeic rabbit. Approximately 0.5 g of intestinal content was diluted to approximately 500 vegetative cells of C. guttulatus/mL with sterilised distilled water. Then, a 20 μL suspension was added to 10 mL YPG (pH 1.5) medium supplemented with 100 mg/L ampicillin. The strain was cultured in a 96-well culture plate with 100 μL of medium per well at 37 °C with 10% CO2 for 2 h. Wells with a single C. guttulatus vegetative cell were further cultured for 5 days before monoclonal cells of C. guttulatus were smeared and cultivated on solid YPG plates (pH 4.5) supplemented with 100 mg/L ampicillin at 37 °C with 10% CO2. A single colony of C. guttulatus was transferred to liquid YPG medium (pH 4.5) and cultivated at 37 °C on an orbital shaker  at a constant rotating speed of 200 rpm. Yeast multiplication was monitored using an automatic microbial growth curve analyser  and an optical density scanner . Culture conditions were optimised by varying the medium pH and culture temperature.A C. guttulatus Zhejiang strain was observed under a light microscope at 400× magnification. Molecular identification was performed by PCR and gene sequencing. Two specific primer pairs for the small subunit (18S) and large subunit (26S) ribosomal RNA genes were synthesised according to Kurtzman C.P. (1998) [C. guttulatus vegetative cells were directly used as the template for PCR. The PCR products were examined and separated by 1% agarose gel electrophoresis. The target bands were purified by a gel extraction kit and sequenced by Sangon Biotech . The sequenced 18S and 26S gene fragments of the C. guttulatus Zhejiang isolates were submitted to GenBank with the Bankit procedure and blasted in the NCBI  database. Similar reference sequences were retrieved. Phylogenetic analysis was performed using the MegAlign program . The phylogenetic tree was constructed using the Clustal W method. Information about C. guttulatus isolates and other yeast species used in the construction of the phylogenetic trees is listed in The morphology of the . (1998) . The prin = 4) were orally inoculated with 1 × 106, 1 × 107 or 1 × 108C. guttulatus vegetative cells per rabbit and designated G1, G2 and G3, respectively. Another group (G4) was not inoculated with yeast and served as the control. Body weight, activities, appetite and excreta were recorded before and after yeast inoculation. Faeces from all groups were collected daily and examined for yeast cells under a light microscope.Three groups of 20-day-old SPF rabbits ; therefore, tissues from different regions of the stomach were immediately frozen for microscopic examination and fixed in 10% formalin for the preparation of paraffin sections for microscopic observation after staining with PAS (periodic acid–Schiff) and in 5% glutaraldehyde for the preparation of electron microscopy sections for observation by transmission electron microscopy.7C. guttulatus cells per rabbit (n = 4). After 14 days, CG/EI and another group  were infected with 1×104 sporulated oocysts of E. intestinalis. One group (designated NON) served as the uninfected control. Body weight, activity, appetite and excreta of animals were recorded before and after inoculation. Faeces were collected for coccidial oocysts and yeast cell counting. E. intestinalis oocysts in faeces were counted between 10 and 16 days after infection using the McMaster method as described previously [C. guttulatus were counted from 2 days before to 14 days after the infection of E. intestinalis. Briefly, 1 g of faeces was mashed with a glass stick and mixed with 60 mL of tap water. The faecal suspension was filtered through a 100-mesh sieve. C. guttulatus cells in the filtrate were counted in a haemocytometer under a light microscope at 100× magnification.Two groups (designated CG and CG/EI) of 28-day-old SPF rabbits were orally inoculated with 4 × 10eviously . VegetatFaecal samples were collected from 253 healthy rabbits in four regions, including Fuyang, Haining, Deqing and Wencheng , in Zhejiang Province, China. The sample numbers from FY, HN, DQ and WC were 50, 50, 49 and 104, respectively. Among the surveyed rabbits, 66 were under 60 days old, and 187 were over 60 days old. The collected samples were stored at 4 °C and examined within one week. The examination was performed as follows: 2 g faeces was mixed with 60 mL of tap water. The mixture was filtered through a 100-mesh sieve. Then, 100 μL filtrate was collected for wet mount examination under a light microscope at 100× magnification. Twenty fields were observed for each sample.C. guttulatus cell counts, rabbit body weight, and E. intestinalis oocyst counts by GraphPad Prism 5.01. Data are expressed as the mean ± standard deviation of four replicates, and experimental groups were compared by using paired-samples t-tests. Differences between groups with p-values < 0.05 were considered statistically significant.Statistical analyses were performed for C. guttulatus is very common in rabbits and is usually not pathogenic even when the dosage is up to 1 × 108 in healthy rabbits; this species seems to be a probiotic microorganism in rabbits, but it may be an opportunistic pathogen when the GI environment is altered by enteric pathogens such as coccidia. Considering the high prevalence of both C. guttulatus and coccidia in rabbits, the potential harm of C. guttulatus to the rabbit industry warrants attention and needs further study.As a commensal yeast,


While chemical Ca2+ indicators served to visualize and measure changes in glial cell cytosolic Ca2+ concentration for several decades, genetically encoded Ca2+ indicators (GECIs) have become state of the art in recent years. Great improvements have been made since the development of the first GECI and a large number of GECIs with different physical properties exist, rendering it difficult to select the optimal Ca2+ indicator. This review discusses some of the most frequently used GECIs and their suitability for glial cell research.Ca GECIs have been employed to study glial Ca2+ signaling and proven superior compared to chemical Ca2+ indicators  from 2+ sensing domain and a single fluorescent protein, whose fluorescence intensity changes when shifting between Ca2+-free and Ca2+-bound states, whereas FRET (Förster or fluorescence resonance energy transfer) GECIs consist of two fluorescent proteins linked by the Ca2+ binding domain  of donor fluorescence is largely devoid of these artifacts but image acquisition is much slower and costly hybrid detectors and photon-counting boards are required. For quantification of Ca2+ signals with FRET sensors, the Ca2+ binding domain used to construct the GECI must also be considered. The Ca2+ binding domain of calmodulin, e.g., binds four calcium ions with positive cooperativity with a Hill coefficient in the range of 2, leading to a non-linear representation of the ambient Ca2+ concentration by the FRET ratio  and multiple Ca2+ binding sites (four for most of the GECIs) and a key reason why expression levels must be kept low.Not only the properties of the fluorescent proteins determine the usefulness of the Cacurrently used for glial cell research or related fields, their key properties, and for which applications they are best suited. As outlined above, there are many factors to consider when selecting the appropriate GECI. There are numerous available GECIs. Alone the derivatives descending from the first GCaMP developed by Nakai et al.  comprise2+ indicators. These must be matched to the light source, detectors, and filter sets available or new optical elements must be purchased. The excitation and emission maxima are listed for the various indicators. Since only the donor fluorophore is excited when using dual fluorophore (FRET) sensors, the excitation spectrum and maximum of the acceptor fluorophore are not quoted but it is highly advisable to also have the ability to directly excite the acceptor to visualize the cells, e.g., in order to select the optimal field of view.The first considerations are the excitation and emission spectra of the Cad reflects the Ca2+ concentration at which half of the Ca2+ indicator molecules are bound by Ca2+, while the other half is dissociated . This is much less important for FRET sensors as one can always excite the acceptor directly to visualize and focus before switching to excite the donor for the actual ratiometric measurements.The GECIs with the highest dynamic range often achieve this by extremely low fluorescence in the apo state, i.e., when no Ca2+-saturated (Fmax) and the fluorescence intensity in Ca2+-free conditions (Fmin) divided by the fluorescence value in Ca2+-free conditions: (Fmax − Fmin)/Fmin. For FRET sensors, it is similarly calculated by using the FRET ratio values instead of the fluorescence values. As mentioned above, while a high dynamic range is always desirable for single-wavelength GECIs it often reflects very low baseline fluorescence rather than very bright Ca2+-bound fluorescence. It is therefore important to consider not only the dynamic range but also how bright the sensor is at baseline. Whereas the best single wavelength sensors now have dynamic ranges of several hundred percent, the FRET sensors always appear inferior in this regard at first glance. In fact, a dynamic range of 40% is excellent for a FRET sensor.For single-fluorophore indicators, the dynamic range is calculated by the difference between the fluorescence intensity when Ca2+ by the indicator molecule is reflected by values such as the half rise time (t1/2 rise) and half decay time (t1/2 decay). If binding and unbinding of Ca2+ occurs much slower than the actual increase and decrease in the Ca2+ concentration, the time course of the Ca2+ signal is distorted. Although it was believed for many years that Ca2+ transients in astrocytes rise and decay at slow rates and hence can readily be recorded with Ca2+ indicators with slow on and off kinetics, more recent studies showed that fast Ca2+ signals in astrocyte microdomains can occur at a time scale of tens of ms , a 543-nm or 568-nm laser (green excitation), deep blue lasers , which are necessary for optimal excitation of cyan fluorophores such as CFP, cerulean or turquoise, are much less common. Therefore, we list lasers suitable for ideal (one-photon) excitation in the figurative summaries. In addition to confocal microscopy, an increasing number of laboratories use two-photon microscopy, in particular, to study glial Ca2+ signaling in vivo  are better options  to 334 nM , and the slowest of these is as fast as GCaMP6f. None rival the apo brightness of jGCaMP7b, although resting brightness will also depend on resting Ca2+ concentration. Also of interest are jYCaMP1 and jYCaMP1s (Kd 79 and 70 nM) and XCaMP-Y, which are particularly suitable for 2-photon excitation using inexpensive pulsed lasers with a fixed wavelength around 1,030 nm (Inoue et al., 2+ indicators designed to retain the excellent responsiveness of the R-GECO series and reduce photoswitching, which limits the usefulness of i.e., jRGECO1a (Shen et al., While we have limited the indicators presented in 2+, FRET sensors are the method of choice, however, transgenic mouse models and ready-to-use viruses are not commercially available. Among the FRET sensors, Twitch-2B is a good choice and has been reported to work in non-excitable cells such as microglial cells (Brawek et al., 2+ concentrations, hence GCaMP6f is favored as it allows visualization of astrocytes at rest. We have recently found that the single-wavelength sensor jGCaMP7b possesses an excellent combination of properties for use in astrocytes, including large dynamic range, fast kinetics, and high resting fluorescence and therefore included it in There is not a single GECI that provides optimal characteristics for all applications. For quantification of changes in cytosolic CaAll authors contributed to the research that is the base for this review as well as to the writing and editing of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In addition to modifying intracellular coupling, the effect of cAMP on insulin secretion could also be at least partly explained by the impact on intercellular coupling. In this review, we systematically describe the possible roles of cAMP at these intra- and inter-cellular signaling nodes, keeping in mind the relevance for the whole organism and translation to humans.Pancreatic beta cells secrete insulin in response to stimulation with glucose and other nutrients, and impaired insulin secretion plays a central role in development of diabetes mellitus. Pharmacological management of diabetes includes various antidiabetic drugs, including incretins. The incretin hormones, glucagon-like peptide-1 and gastric inhibitory polypeptide, potentiate glucose-stimulated insulin secretion by binding to G protein-coupled receptors, resulting in stimulation of adenylate cyclase and production of the secondary messenger cAMP, which exerts its intracellular effects through activation of protein kinase A or the guanine nucleotide exchange protein 2A. The molecular mechanisms behind these two downstream signaling arms are still not fully elucidated and involve many steps in the stimulus–secretion coupling cascade, ranging from the proximal regulation of ion channel activity to the central Ca The decrease in K+ efflux causes membrane depolarization, followed by the opening of voltage-dependent Ca2+ (VDCC) channels and the influx of Ca2+ ions. The resulting increase in the intracellular concentration of Ca2+ ([Ca2+]IC) triggers insulin secretion by activating the secretory machinery and fusion of insulin-containing vesicles with the plasma membrane IC [Ni et al. found that oscillations in PKA activity directly mirror the oscillations in cAMP levels . The cAM2+]IC .2+-sensitive AC into nanodomains is then responsible for local increases in cAMP levels, which are exactly in-phase with Ca2+ oscillations, whereas localized PDE is responsible for the out-of-phase decreases in local cAMP ) [ATP]IC/[ADP]IC [Kchannels . They ar/[ADP]IC ,81.ATP channels in an ADP-dependent manner IC  a leftward shift in voltage dependent activation and (ii) rightward shift in voltage dependent steady-state inactivation. A leftward shift in voltage dependent activation means that the maximal current through the channels is recorded at a lower membrane potential when GLP-1 is added. This is likely mediated by a PKA-induced phosphorylation of the channels. Due to a rightward shift in steady-state inactivation curve, fewer channels are inactivated at a given membrane potential and are therefore ready to be activated once again at a higher membrane potential [The effect of GLP-1 on [Ca2+]IC B was rececretion ,41, and ntration ,117,118.orskolin ,121,122.otential ,124.v1.3 seems to be preferentially coupled to GLP-1 receptor activation. This signaling pathway depends on intact intracellular stores, PKA and PKC activation  but did not induce CICR. The addition of exendin-4 failed to induce [Ca2+]IC increase in beta cells before uncaging of Ca2+ by UV flash photolysis, indicating the effect of exendin-4 in this experiment was not mediated through membrane depolarization and subsequent opening of VDCC [2+ release mechanism to the stimulatory effect of an increase in [Ca2+]IC, thereby allowing CICR to be triggered by uncaging of Ca2+ IC. There is a report indicating that Epac2A activators might signal through nicotinic acid adenine dinucleotide phosphate (NAADP) receptors, known also as two-pore Ca2+ channels, to mobilize Ca2+ from non-ER compartments. These acidic stores are presumed to be responsible for the fast, transient phase of Ca2+ response IC oscillations and other types of cellular responses, but a detailed characterization of changes in [Ca2+]IC dynamics during metabolic amplification by cAMP is lacking in published literature. The qualitative and the quantitate diversity of results arise from the variety of methods and protocols used to investigate Ca2+ responses in pancreatic beta cells.cAMP also affects [Ca2+]IC oscillations rely on periodic entry of Ca2+, the fast pattern also seems to depend on mobilization of intracellular Ca2+ stores, and agents that increase cAMP promote the appearance of regular fast [Ca2+]c oscillations IC peak, occasionally followed by a smaller, sustained [Ca2+]IC elevation [2+]IC dynamics and proposed a mechanism behind them. The first one is transient on the timescale of seconds and is believed to be due to CICR activated by a Ca2+ influx trough L-type VDCCs. The second, more sustained Ca2+ rise measured in minutes reflects an increased flux of Ca2+ ions through plasma membrane through L-type VDCCs as well as through nonselective cation channels. These fast Ca2+ transients were recorded in beta cells voltage-clamped at 50 mV, a membrane potential above the resting potential, to allow GLP-1 to promote the opening of VDCCs, and the rise in Ca2+ was not associated with a change in membrane current. However, the fast transients were blocked by nimodipine, exposure to a Ca2+-free medium, hypoglycemic glucose concentrations, thapsigargin, and ryanodine, all supporting the model where GLP-1 facilitates Ca2+ influx and RYR-mediated CICR to amplify insulin granule exocytosis IC oscillations are recorded in response to glucose stimulation, addition of GLP-1 or cAMP agonists typically increases their frequency IC, which is sometimes preceded by an escalation in oscillatory frequency IC elevation with attenuation or abolition of Ca2+ oscillations [2+]IC. The older studies of Bode et al. and Flamez et al. used Fura-2 and Fure-PE3/AM, respectively, capturing emitted fluorescence by a CCD camera. Contemporary Ca2+ indicators that are compatible with confocal microscopy and have an improved sensitivity to rapid changes in Ca2+ dynamics offer enhanced insight into beta cell dynamics that was previously inaccessible IC are minor, the amount of insulin secreted in response to cAMP elevation is much greater, indicating that the rise in [Ca2+]IC is just one and probably not the most quantitatively important cAMP-sensitive step in reference IC in the SSC cascade, cAMP also directly affects exocytosis (− influx . F. F2+]IC ocytosis C 86]. E. E2+]IC − influx . Epac2A − influx ,160, at − influx . Epac2A − influx . PKA-depflux IC oscillations, increased GSIS, and reduction in beta cell coupling [2+]IC and exocytotic responses were enhanced IC oscillations, sensitivity of the exocytotic machinery, or intercellular coupling during diet-induced T2DM. A single study demonstrated that, in human islets, cAMP stimulates synchronous [Ca2+]IC increases that augment insulin secretion, and the signals become asynchronous with an accompanying reduction in insulin secretion in lipotoxic conditions IC changes, to the most distal event, exocytosis. We also addressed the aspect of intercellular coupling and the relevance in vivo. It is quite self-evident that our knowledge about the role of cAMP is limited and biased due to the influence of historically and currently available electro- and optophysiological tools, pharmacological agents, development of specific animal models, and access to human tissue, among many other factors. Since a deeper insight into the normal intra- and intercellular coupling in beta cells and the role played by cAMP is central for a better understanding of the natural history of diabetes mellitus as well as for prevention, finding new targets, and developing new pharmacological approaches, we firmly believe that future studies will help push the frontiers of knowledge and truly hope that our review helped identify some most needed next steps.Concentrating on a possible role of cAMP in general and its two main signaling arms in specific, our journey led us from the most proximal membrane potential step in the SSC cascade, via the central [Ca