Cultured NHLF were loaded with Fura-2 AM (3 μM) and Ca2+ signals were recorded by microfluorimetric techniques. Beractant causes a concentration-dependent increase in [Ca2+]i with a EC50 of 0.82 μg/ml. The application of beractant, at a concentration of 500 μg/ml, which has been shown to exert an apoptotic effect in human fibroblasts, elicited different patterns of Ca2+ signals in NHLF: a) a single Ca2+ spike which could be followed by b) Ca2+ oscillations, c) a sustained Ca2+ plateau or d) a sustained plateau overlapped by Ca2+ oscillations. The amplitude and pattern of Ca2+ transients evoked by beractant were dependent on the resting [Ca2+]i. Pharmacological manipulation revealed that beractant activates a Ca2+ signal through Ca2+ release from intracellular stores mediated by phospholipase Cβ (PLCβ), Ca2+ release from inositol 1,4,5-trisphosphate receptors (IP3Rs) and Ca2+ influx via a store-operated pathway. Moreover, beractant-induced Ca2+ release was abolished by preventing membrane depolarization upon removal of extracellular Na+ and Ca2+. Finally, the inhibition of store-operated channels prevented beractant-induced NHLF apoptosis and downregulation of α1(I) procollagen expression. Therefore, beractant utilizes SOCE to exert its pro-apoptotic and antifibrinogenic effect on NHLF.Beractant, a natural surfactant, induces an antifibrogenic phenotype and apoptosis in normal human lung fibroblasts (NHLF). As intracellular Ca Pulmonary surfactant is a liquid layer covering the alveolar network of mammalian lungs and composed of approximately 90% lipids (mainly phospholipids) and 10% proteins (mainly surfactant-associated proteins or SAPs) ; in addiIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive and lethal lung disorder, as patients show a median survival of 3–5 years after diagnosis . IPF is Studies on the effect of surfactant components on non immune cells are scarce. In lung fibroblasts, it has been shown that beractant, an exogenous lung surfactant replacement preparation, downregulates DNA synthesis and inhibits interleukin-1 (IL-1)-stimulated secretion of IL-6 and prostaglandin E2 . Likewis2+ signaling is implicated in apoptosis [2+ signal in NHLF. An increase in intracellular Ca2+ concentration ([Ca2+]i) can be caused either by Ca2+ entry from the extracellular milieu or by Ca2+ release from internal storage compartments i i. Ca2+ influx is an ubiquitous event that occurs through a number of distinct membrane Ca2+-permeable pathways, including voltage-operated, receptor-operated, second messenger-operated and store-operated Ca2+ channels (SOCs) i . While Cs (SOCs) –202+ homeostasis in NHLF. Accordingly, we aimed to assess the effect of beractant on [Ca2+]i in primary cultures of NHLF, by using conventional imaging microscopy. Our results showed that beractant induces a concentration-dependent Ca2+ signal by the concerted activation of PLCβ, Ca2+ release from IP3Rs and store-operated Ca2+ entry (SOCE). The pharmacological blockade of SOCE, in turn, prevented the functional effects of beractant on NHLF, i.e. induction of apoptosis and downregulation of α1(I) procollagen expression. Therefore, SOCE is the most likely candidate to mediate the effects of beractant on NHLF.It is currently unknown whether beractant alters Ca2 and 5% CO2. Only cells grown from lungs with normal histology were considered for this study. Fibroblasts from passages 5–10 were plated onto coverslips placed in Petri dishes. Cells were allowed to attach to the coverslips for 24 hours, and then incubated for 48 hours in serum free medium.Primary NHLF were obtained in our laboratory as previously described . Briefly2+]i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (shortly termed "Ratio (F340/F380)”. An increase in [Ca2+]i causes an increase in the Ratio (F340/F380). Ratio measurements were performed and plotted on-line every 3 s. Images were stored on the hard disk and converted offline to 340/380 ratio images by ImageJ software . The experiments were performed at room temperature (21–23°C).NHLF attached to the coverslips were washed twice with physiological salt solution (PSS) and loaded with 3 μM Fura-2 acetoxymethyl ester in PSS for 30 min at room temperature. The cells were incubated for 30 min in PSS free of Fura-2. The coverslips were washed and fixed to the bottom of a Petri dish using silicone grease. The Petri dish was mounted onto the stage of an upright epifluorescence Axiolab microscope , equipped with a 100-W mercury lamp. A Zeiss X63 Achroplan objective  was used to visualize the cells. NHLF were excited alternately at 340 and 380 nm, and the emitted light was detected at 510 nm. A neutral density filter  was coupled to the 380 nm filter to approach the intensity of the 340 nm light. A round diaphragm was used to increase the contrast. The exciting filters were mounted on a filter wheel equipped with a shutter . Custom software, working in the LINUX environment, was used to drive the camera  and the filter wheel, and to measure and plot on-line the fluorescence from a number of 6–10 rectangular “regions of interest” (ROI) enclosing 6–10 single cells. Each ROI was identified by a number. phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2) was purchased from Calbiochem . Ham’s F-12 medium and FBS were purchased from GIBCO BRL, Life Technologies, Grand Island, NY. All other chemicals were purchased from Sigma-Aldrich.2+ peak and plateau amplitude (ΔR) were normalized to resting fluorescence (Ri) to compare the height of the Ca2+ responses to beractant produced by cells displaying different basal fluorescence levels (ΔR/Ri). Mean values are presented together with standard error of the mean and the number “n” of tested cells. Statistical comparisons of peak amplitudes were made by Student’s t-test, p<0.05 was considered significant. As to the plateau phase, the number x of cells responding to the experimental test over the number y of tested cells (x/y) is usually reported. Unless differently stated, tracings shown in the figures are single ROI recordings.For each protocol, data were collected from NHLF isolated from lungs of at least three healthy donors. The amplitude of the peak response was measured as the difference between the ratio at the peak and the mean ratio of 5-min baseline before the peak. Such a difference was considered as a physiological signal when it was > 2 times the SD of the baseline. Ca2+ transient amplitude), [Beractant] is the beractant concentration, and half-maximal effective concentration (EC50) is the [Beractant] that caused 50% of the response of control.The beractant concentration-response data were fit to an equation of the form:-ΔΔCt).Real time quantitative polymerase chain reaction was used to measure the expression of collagen in human fibroblasts after 24 hours of incubation. Total cell RNA was extracted with TRIzol reagent  as per the manufacturer's instructions. RNA was reverse transcribed into cDNA and real-time PCR was performed in the Step One Real-Time PCR System  with TaqMan probe labeled with FAM (Hs00164004_m1 for _1-type-I collagen). Gene target levels in each sample were normalized against GAPDH as internal control. Cycling conditions were 2 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C concluding with an infinite loop of refrigeration. All real time PCRs were performed in triplicate at least two times. Results were normalized to human GAPDH according to the delta-delta Ct method . Each well of a 96 well/culture plate contained 10,000 fibroblasts in Ham F12 medium; the plate was incubated at room temperature with 100 μl Caspase-Glo reagent for thirty minutes. A blank reaction was included which only contained cell culture medium without cells. The luminescence of each sample was measured in a plate-reading luminometer .2+ signal in 96.27% of Fura-2-loaded cells (568 out of 590 cells). Beractant elicited a heterogeneous pattern of Ca2+ signals even in cells from the same microscopic field  induced a biphasic Ca2+ response in all the cells analyzed (n = 28)  .2+]i simultaneously in many individual NHLF from the same population. The mean value of basal [Ca2+]i measured in all cells studied in the present work was 0.463±0.009 ratio arbitrary units (A.U.) (n = 590). In the majority of cells which had a resting [Ca2+]i higher than 0.56 ratio A.U , or repetitive Ca2+ oscillations (n = 75) , as related to those that had levels of basal [Ca2+]i lower than 0.4 ratio A.U. (i.e. plateau and plateau + oscillations), as summarized in 2+ spike was significantly (p<0.05) higher in plateauing cells as compared to single-spiking and oscillating cells . The same results were obtained when the Ca2+ response to beractant consisted in the onset of repetitive Ca2+ oscillations (2+ transient (13.46%), but the decrease was not statistically significant .Beractant effect in NHLF was reversible: the Callations , n = 45.2+ signals enabled us to establish the concentration-response relationship by the repeated administration of the agonist to the same cells. The application of increasing concentrations of beractant (0.03–500 μg/ml) to Fura-2 loaded NHLF produced a concentration-dependent increase in [Ca2+]i. 2+ increases in response to beractant (0.1 to 500 μg/ml) in a NHLF that presented a single spike pattern response (see 2+ response (not shown). The non-cumulative concentration-response curve of beractant-induced elevation in [Ca2+]i is depicted in EC50), calculated by fitting the concentration-response curve as described in Materials and Methods, was 0.82 μg/ml. Notably the R2 value for the curve fit was 0.9928  to prevent Ca2+ entry through the plasma membrane. Beractant caused an immediate increase in [Ca2+]i in the absence of extracellular Ca2+ in 48 out of 50 cells, although both the Ca2+ oscillations and the plateau phase disappeared  as compared to the Ca2+ transient evoked by beractant in presence of extracellular Ca2+ (see statistics in 2+ vs Beractant 2nd). These results indicate that the peak response is due to both Ca2+ influx and Ca2+ release, whereas Ca2+ entry sustains both the plateau phase and the following oscillations in [Ca2+]i. This notion is corroborated by the experiment depicted in 2+ reversibly inhibited the plateau in 28 of 28 cells    ; CPA is epletion –22. In C(n = 10) .2+ signals was studied by preincubating the cells with U73122 (10 μM), a widely employed PLC inhibitor i that may follow the initial Ca2+ spike triggered by beractant  as well. In order to assess the hypothesis that the sustained Ca2+ signal evoked by beractant was mediated by VOCC in NHLF, we probed the effects of Ni2+ (100 μM), a non specific blocker of VOCC, and nifedipine (1 μM), which selectively antagonizes L-type VOCC. Neither Ni2+  i in 21 of 21 NHLF cells and could not be utilized further (2+ channels i in NHLF, which might be involved in its functional effect on these cells.Restoration of surfactant activity has been introduced in the routine care of patients affected by respiratory distress syndrome , and migntiation ,33,40. I2+]i in neighboring NHLF, which displayed at least 4 types of responses upon agonist stimulation: 1) a rapid Ca2+ spike which quickly decayed to the baseline; 2) a biphasic Ca2+ signal, which comprised an initial Ca2+ spike followed by a prolonged plateau phase of intermediate amplitude; 3) repetitive oscillations in [Ca2+]i and 4) a biphasic elevation in [Ca2+]i featured by the superimposition of Ca2+ oscillations on the plateau phase. Studies of the Ca2+ responses of a wide variety of cell types at the single cell level have consistently revealed cell-to-cell heterogeneity i elevations elicited by bradykinin [2+ response to EGF [2+]i when exposed to beractant and that this is not due to cell heterogeneity in the cell cycle.Beractant evoked a complex pattern of elevations in i in NHLF might invoke single-cell heterogeneity regarding membrane receptors or elements of the phosphoinositide signaling pathways, such as PLCβ, IP3Rs, and SOCE (see below), as recently suggested in [2+ levels: The basal [Ca2+]i is higher in cells displaying either a single Ca2+ spike or discrete Ca2+ oscillations as compared to those experiencing the plateau phase, with or without the superimposition of sinusoidal oscillations. Similar results were found by Toescu and coworkers i > 150 nM i signal, the so-called Ca2+ fingerprint i is equal to 0.82 μg/ml, while its maximal effect was achieved at 100–500 μg/ml. Likewise, in NHLF, beractant was found to induce apoptosis and reduce collagen deposition at 500 μg/ml i is prevented by U73122, a widely employed PLC inhibitor, while it is unaffected by its structural analogue, U73343. genistein, a broad spectrum protein tyrosin kinase inhibitor, did not interfere with the onset of the Ca2+ response to beractant. Therefore, PLC activity is likely to be induced by the activation of GPCRs and to involve the β-isoform. Second, beractant-induced Ca2+ signals are prevented by 2-APB, a membrane-permeable blocker of InsP3-dependent Ca2+ release in the absence of external Ca2+. Third, no Ca2+ signal could be detected in response to caffeine, which stimulates endogenous RyRs by sensitizing them to resting Ca2+ levels i. Fifth, lanthanides reversibly abrogated the sustained component of the Ca2+ response to beractant, by interrupting both the prolonged plateau phase and the repetitive Ca2+ oscillations. This effect was observed when both La3+ and Gd3+ were applied at 10 μM, a concentration which selectively hinders SOCs [2+]i in NHLF. These findings strongly indicate that, while the single Ca2+ transients exclusively derive from IP3-dependent Ca2+ mobilization, the sustained response involve Ca2+ entry through plasmalemmal SOCs. This concept is corroborated by the finding that, when 2-APB is administrated in the presence of extracellular Ca2+ to interfere with IP3Rs and SOCE, both the prolonged plateau phase and the repetitive Ca2+ spikes rapidly run down. According to the most popular models proposed to describe intracellular Ca2+ oscillations  lower and higher than 10 μM, respectively i in neutrophils due to G protein-mediated release from intracellular Ca2+ stores. Beractant-induced InsP3-dependent Ca2+ mobilization requires the insertion of monovalent cationic channels by SAP-B and SAP-C, which depolarize the cells by causing Na+ influx i. The mechanistic link between the positive shift in membrane potential and PLCβ activation is likely to be provided by voltage-dependent GPCRs, such as P2Y1, 5HT2A, thromboxane (TPα), M1 and M3 receptors i, such as that produced by a sustained plateau, is amid the most powerful apoptogenic signals [3+ and 2-APB prevented beractant-induced NHLF apoptosis. On the other hand, intracellular Ca2+ oscillations encode the information driving the Ca2+-dependent activation of several transcription factors [3+ prevented beractant from suppressing the downregulation of α1(I) procollagen transcript. 2-APB reduced α1(I) procollagen expression per se and could not be used further, but we should recall that this drug is far less selective than 10 μM Gd3+ and interferes also with other Ca2+-permeable pathways [2+ signaling induced by beractant in NHLF control different cellular processes is currently under evaluation.After lung injury, recovery depends on reestablishment of the air-lung interface through the elimination of intra-alveolar mesenchymal cells. Beractant induces apoptosis and decreases collagen accumulation in NHLF ; accordi signals . Accordi factors ,65. Intrpathways . The hyp2+ signals elicited by a natural lung surfactant extract in primary cultures of human lung fibroblasts. The Ca2+ response to beractant is triggered by PLCβ recruitment following the activation of a GPCR. The subsequent cleavage of the membrane phospholipid, PIP2, leads to the generation of IP3, which releases intraluminally stored Ca2+, thereby activating SOCE. The interplay between IP3-dependent Ca2+ mobilization and SOCE results in a variety of Ca2+ signals depending on the resting Ca2+ levels. Beractant-induced Ca2+ signals might protect against pulmonary structural remodeling in IPF as well as other severe fibrosing respiratory diseases.In summary, our results describe for the first time the pattern of CaS1 FigA) Ca2+ response to ATP (100 μM) in NHLF. ATP-induced Ca2+ elevation was abrogated by B) depletion of intracellular Ca2+ stores with CPA (10 μM) in absence of extracellular Ca2+ (0Ca2+), and C) by blockage of PLC activity with U73122 (10 μM).(TIF)Click here for additional data file.S2 Fig2+ signal evoked by the pyrazole derivative, BTP-2 (20 μM), in a single NHLF cell.Ca(TIF)Click here for additional data file.


In a previous study, we demonstrated SOCE activity in intact mouse cardiac pacemaker tissue, specifically from sinoatrial node (SAN) tissue. However, store content as a key determinant of SOCE activity is difficult to measure in intact SAN tissue. Therefore, to investigate the interaction between SOCE and store content and its role in cardiac pacemaking, it is necessary to investigate SOCE activity in single cardiac pacemaker cells. Furthermore, recent studies in other tissues have identified two new proteins involved in SOCE, stromal interacting molecule (STIM), which is an ER Ca2+ sensor, and the surface membrane channel Orai, a prototypic gene encoding for SOCE. However, whether STIM and Orai are expressed in native pacemaker cells is still unknown. In this current study, we examined SOCE activity in single firing pacemaker cells isolated from mouse sinoatrial node tissue. We found a significant rise in Ca2+ entry in response to Ca2+ store depletion. SOCE blockers reduced the amplitude and frequency of spontaneous Ca2+ transients and reduced Ca2+ store content. We demonstrated for the first time that STIM and Orai are expressed in pacemaker cells. After store depletion, STIM1 redistributed to the cell periphery and showed increased co-localization with surface membrane located Orai1, indicating a possible involvement of these proteins in SOCE activity in native cardiac pacemaker cells. These results suggest the novel concept that SOCE plays a functional role in regulating intracellular Ca2+ of cardiac pacemaker cells.In many non-excitable and excitable cells, store-operated calcium entry (SOCE) represents an additional pathway for calcium entry upon Ca The loss of Ca2+ from the SR causes partial depletion which recovers as the SR Ca2+ pump (SERCA) returns Ca2+ to the SR. Ca2+ extrusion through the Na+/Ca2+ exchanger generates an inward current that contributes to pacemaker diastolic depolarization.  of the heart. Originally it was thought that the spontaneous firing of pacemaker cells was driven purely by voltage-dependent membrane currents Noble,  but subs2+ concentration within the lumen of the endoplasmic reticulum (ER) or SR. Activation of SOCE produces Ca2+ influx which provides a regulatory link between SR Ca2+ level and membrane calcium entry, thereby helping to maintain SR calcium homeostasis  when extracellular Ca2+ was replenished after store depletion  represents an additional pathway for calcium entry into the cell to refill the SR calcium store. SOCE is activated by the decline of Ca2+ in response to SR Ca2+ store depletion. Using two different blockers of SOCE, SKF96365, and BTP-2, we estimated the contribution of SOCE to pacemaker activity and the refilling of the SR Ca2+ store.In the present study, we examined SOCE in single firing pacemaker cells, by measuring the changes in intracellular Ca2+ influx through TRPC channels has also been implicated in cardiac hypertrophy     under a protocol approved by the Animal Ethics Committee of the University of Sydney.2+ using a dissecting microscope. A strip of tissue containing the SAN region, measuring ~0.5 mm × ~1 mm, was identified by anatomic landmarks and dissected out. The SAN strips were cut into 3–5 smaller strips and rinsed in a “Ca2+ free” solution containing (in mmol/L) 120 NaCl, 5.4 KCl, 0.5 MgCl2, 1.2 KH2PO4, 20 taurine, 11.1 glucose, 10 HEPES, 0.3 EGTA, 10 Na-Pyruvic acid, 5 Creatine, 5 μmol/L Blebbistatin, 2 mg/ml bovine serum albumin (BSA), pH 7.0. The rinsed SAN tissue strips were digested in the same “Ca2+ free” solution containing collagenase , elastase (1.9 u/ml) and protease  for 30–40 min at 35 ± 0.5°C and bubbled with pure oxygen. After enzyme digestion, the tissue was then washed and stored in Kruftbrühe (KB) solution which contains (in mmol/L), 30 KCl, 10 KH2PO4, 2 MgCl2, 70 L-glutamic acid, 10 HEPES, 20 taurine, 5 creatine, 0.3 EGTA, 10 Na-Pyruvic acid, 5 Creatine, 5 μmol/L Blebbistatin, 80 KOH, 11.1 glucose and 10 HEPES, with pH adjusted to 7.2 with KOH. Single SAN cells were released by gentle pipetting of the digested tissue strips. SAN cells were identified under light microscopy by their spindle shape and small size with centered single nuclei. Atrial myocytes were dissociated following the same isolation protocol while ventricular myocytes were dissociated by the collagenase-based coronary perfusion method as described elsewhere  for 20 min to de-esterify Fluo-4-AM, spontaneously beating pacemaker cells were selected for study. A LSM 510 META confocal microscope  equipped with an argon laser provided excitation at 488 nm and the fluorescence signal was collected at wavelengths of >515 nm. A heated microscope stage and a 63 × /1.4 oil objective heater  were attached to maintain cells at 37°C throughout the experiment. Cell shortening was recorded by line scan mode and analyzed offline with ImageJ software . Intracellular Ca2+ was recorded using either line scan mode (xt mode) or frame mode (xy mode) with time series. Results were recorded at Zeiss LSM scan speed 8 (3.07 ms/line) and 10 s intervals exist between two consecutive frames in frame mode. Data were analyzed with Zeiss LSM image examiner  and imageJ . Global intracellular Ca2+ levels were translated into relative fluorescence levels, F/F0, where F and F0 represent the fluorescence intensities at a given time and at minimum resting level, respectively. Maximum change in F/F0 (in frame mode) was calculated as the change in the peak value after Ca2+ re-admission relative to initial level before Ca2+ removal.Isolated single mouse SAN cells were placed on laminin-coated (20 μg/ml) 35 mm glass bottom petri dishes (MatTek Cultureware) for 20 min to attach. Fluo-4-AM  was mixed with Pluronic F-127 by sonication and diluted to a final concentration of 5 μmol/L in Tyrode solution. This solution was used to load single pacemaker cells (20 min at room temperature). After superfusion with normal Tyrode solution  and spleen cells were extracted with TRIzol (Invitrogen. USA) following manufacturer's protocol. Reverse transcription was carried out with 1 μg total RNA using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen), according to the manufacturer's instructions. RT-PCR was performed with Platinum Taq (Invitrogen) under the following conditions as described by Wissenbach et al. : one cyc2+ free Tyrode Solution with either 5 μmol/L cyclopiazonic acid (CPA) or 1 μmol/L thapsigargin (TG) for 30 min at room temperature before fixation. Fixed cells were washed three times with phosphate buffer solution (PBS) over 30 min, permeabilized by 0.1% Triton X-100 (Sigma) for 5 min, washed three times with PBS over 30 min, and blocked with 1% bovine serum albumin  and 4% normal Goat serum  in PBS for 1–2 h before application of primary antibody. Primary antibodies were diluted in 1% BSA and 4% normal goat serum in PBS. Cells were incubated with primary antibodies at an appropriate concentration , atrium  and ventricle  were freshly dissected and snap frozen in liquid nitrogen. Spleen tissue was collected in the same way and served as positive control protein. Total protein extracts were prepared with Mammalian Cell Lysis Kit  containing RIPA buffer and protease inhibitor cocktail. Tissue samples were homogenized on ice using a Polytron homogenizer (PT900 CL) and cleared by centrifugation at 12,000 g for 10 min. Protein extracts were separated on 8% SDS-PAGE gel and transferred to a nitrocellulose membrane (Whatman). The membranes were immunoblotted with the appropriate antibodies Table . Immunob2+-ATPase (SERCA) inhibitors were used to deplete store content. The imidazole compound SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole hydrochloride) has been shown to target the STIM1-Orai1 pathway and inhibit SOCE in cell lines  and Cyclopiazonic acid  which are SR Cat-test or ANOVA and a value of P < 0.05 was considered statistically significant.Data were presented as means ± SEM. The statistical significance of effects was evaluated by Student's 2+]i in response to an intervention protocol which involves removing extracellular Ca2+ in addition to the application of SR Ca2+-ATPase (SERCA) inhibitor. We previously showed that this protocol caused activation of SOCE in intact mouse SAN tissue , the cell exhibited spontaneous firing and regular Ca2+ transients Figure 2+ withdrawal from the solution for 5 min, which was associated with a significantly reduced [Ca2+]i [Figure 2+]i upon Ca2+ re-admission is demonstrated in Figure 2+]i rapid declined after an initial transient overshoot [Figure 2+ transients associated with pacemaker firing reappeared as [Ca2+]i gradually returned to control level after wash off of CPA [Figure 2+ fluorescence intensity changes deduced from 60 consecutively collected Ca2+ images in response to the SOCE activation protocol. The top panel showed the timing of extracellular [Ca2+] withdrawal and readmission. A large Ca2+ influx reached its maximum within 30 s after Ca2+ re-admission and lasted for 1–2 min  and extracellular Ca2+ free plus thapsigargin (green line). SR Ca2+ store content indicated by caffeine induced Ca2+ transients (quantified by maximum ΔF/F0) showed a small but non-significant reduction after extracellular Ca2+ removal  but exhibited a large and significant fall when extracellular Ca2+ removal was combined with SERCA inhibition . These data confirm that the combination of extracellular Ca2+ removal and application of a SERCA blocker cause store depletion. The large Ca2+ influx is dependent on SR Ca2+ store content and is store-operated Ca2+ entry.To confirm that the large Ca+i rise wnt Bers, . Caffein2+ store   Figure . These d2+ current, ICRAC  or is dependent on G-protein coupled receptor activation (ROCE)  and STIM1 (green). Orai1 labeling appears closer to the cell membrane, while STIM1 labeling also appears along the surface membrane where Orai1 is located. The colocalization of two proteins is demonstrated by the yellow staining as showed in an enlarged panel (arrows) [Figure 2+ 1.8 mmol/L) whereas the store depleted cells were kept in Ca2+ free solution containing the SERCA blockers CPA or TG for 30 min. The translocation after store depletion was quantified by dividing the diameter of cells into 4 equal regions , indicating very little concentration in the cell membrane, but changed significantly to 4.16 ± 0.56 (n = 9 in the presence of CPA) or 4.35 ± 1.38 (n = 9 in the presence of TG) (P < 0.05) when the store was depleted . Similar results were obtained for the colocalization of Orai1 with STIM1 (not shown). This increased colocalization resulting from the peripheral redistribution of STIM1 is expected to increase the opportunity for physical interaction between STIM1 and Orai1, resulting in activation of calcium influx through Orai1.Store-dependent translocation of STIM1 appears to be a prerequisite for the physical interaction with Orai1, which is followed by Orai1 activation and Cas Figure  and the s Figure . For Orad Figure . This daIn the present study, we examined both the functional consequences of SOCE and molecular components of SOCE in isolated pacemaker cells. The study strengthens our earlier identification of SOCE in intact mouse SAN  in firing rate when SOCE was blocked, and thus supports a possible role of SOCE in regulating the firing rate under physiological conditions. Using computer simulation, we recently found that an additional inward SOCE current with a long time constant of activation  could lead to a small increase (~11%) in firing rate. With such a long activation time constant, SOCE current effectively becomes a background current whose level changes little over the cardiac cycle and whose magnitude depends on the mean level of Ca2+ release activated current (ICRAC).In the present study we demonstrate that SOCE activity is present in single pacemaker cells. We also demonstrate that several new SOCE proteins, including STIM1, STIM2, Orai1, and Orai3, but not Orai2, are expressed in pacemaker cells of mouse SAN tissue. Using immunohistochemistry, we show that while Orai1 proteins are predominantly located in the sarcolemma, STIM1 proteins under control conditions are distributed across the cell. It has been reported that STIM activation induces conformational changes of Orai proteins and subsequent STIM-Orai colocalization, which forms the active store-operated calcium channel Lewis, . We alsoWhile the molecular mechanism of STIM1/Orai1 activity and their functional importance have been studied in great detail, the functional relevance of other isoforms of STIM and Orai still remain speculative  (Hofmann et al., In our earlier work we identified that TRPC1, 3, 4, and 6 are all expressed in pacemaker cells (Ju et al., A patch clamp study is needed to further establish whether SOCE is accompanied by an inward current (Potier et al., 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.


Here, the current knowledge about the molecular mechanisms of vacuolar Ca2+ release and Ca2+ uptake is summarized, and how different vacuolar Ca2+ channels and Ca2+ pumps may contribute to Ca2+ signaling in plant cells is discussed. To provide a phylogenetic perspective, the distribution of potential vacuolar Ca2+ transporters is compared for different clades of photosynthetic eukaryotes. There are several candidates for vacuolar Ca2+ channels that could elicit cytosolic [Ca2+] transients. Typical second messengers, such as InsP3 and cADPR, seem to trigger vacuolar Ca2+ release, but the molecular mechanism of this Ca2+ release still awaits elucidation. Some vacuolar Ca2+ channels have been identified on a molecular level, the voltage-dependent SV/TPC1 channel, and recently two cyclic-nucleotide-gated cation channels. However, their function in Ca2+ signaling still has to be demonstrated. Ca2+ pumps in addition to establishing long-term Ca2+ homeostasis can shape cytosolic [Ca2+] transients by limiting their amplitude and duration, and may thus affect Ca2+ signaling.The vacuole is by far the largest intracellular Ca It is reasonable to assume that this huge Ca2+ store contributes to changes in cytosolic free Ca2+ concentrations, [Ca2+]cyt, during Ca2+-mediated intracellular signaling. Usually, the volume of the large central vacuole is about an order of magnitude larger compared to the volume of the cytosol, and the free Ca2+ concentration is about three orders of magnitude higher inside the vacuole, compared to the cytosol. The cytosolic free Ca2+ concentration at rest, as recorded with ion-selective microelectrodes or fluorescent dyes, is around 200 nM, and may increase to low micromolar concentrations during transient [Ca2+]cyt increase cyt, visualized by digital ratio imaging of fluorescent Ca2+ indicators, has been reported to be more pronounced close to the vacuole [2+]cyt triggered by high external Ca2+ concentrations cyt spikes were induced by cooling cyt. Increase of cytosolic InsP3 by release from a caged photoactivatable derivative [2+]cyt. However, in plant cells the molecular mechanisms of InsP3-induced [Ca2+]cyt increase are not well understood, since the typical InsP3 receptor Ca2+ channel found in animals seems to be missing in land plants (Embryophyta). None of the more than 50 sequenced land plant genomes seems to encode an ortholog of the InsP3 receptor  or Trebouxiophyceae  roots cyt increase and stomatal closure might be mediated by myo-inositol hexakisphosphate  [6 in broad bean guard cells resulted in a transient increase in [Ca2+]cyt [6 to isolated broad bean guard cell vacuoles increased whole-vacuole current amplitudes [6-dependent vacuolar currents are different from vacuolar currents induced by 1 μM InsP3 [6-fold [Ca2+] gradient (10 mM in the vacuole and ≈2 nM outside), InsP6-dependent vacuolar currents did not show strong rectification, indicating that ions other than Ca2+ were conducted, and current reversal potentials were close to 0 mV, indicating a lack of Ca2+-selectivity. Therefore, there seems to be no electrophysiological evidence for Ca2+ mobilization by InsP6 from the large central vacuole.Studies with guard cells from potato (phytate) . CorroboCa2+]cyt . Applicaplitudes . These IμM InsP3 ,59,60. E+ by ADP-ribosyl cyclases and functions as a Ca2+ mobilizing second messenger in different eukaryotic clades , which is known to activate ryanodine receptors cyt oscillations were reported to be the result of circadian oscillations of cytosolic cADPR concentrations [2+]cyt oscillations in A. thaliana by InsP3 has been reported as well [2+ release by cADPR seems to be an essential step of ABA signal transduction [2+]cyt resulting in potassium salt release and guard cell turgor loss cyt (≈10 nM) and decreased at increasing [Ca2+]cyt, almost disappearing at 1 μM cyt [2+]cyt might explain why recordings with Ca2+-selective microelectrodes in media containing [Ca2+] ≈ 2 μM failed to detect a cADPR-dependent Ca2+ efflux from isolated vacuoles cyt ,70. The vacuoles . A more 2+ regulated, non-selective cation channel—the slow vacuolar (SV) channel cyt increases the open probability and postulated Ca2+-induced Ca2+ release by the SV channel. In animal cells, an initial small [Ca2+]cyt increase is amplified via Ca2+-induced Ca2+ release by ER ryanodine and InsP3 receptor Ca2+ channels , vacuolar [Ca2+], and trans-tonoplast electrical potential seemed to exclude Ca2+-induced Ca2+ release. As long as the electrochemical potential gradient for Ca2+, ΔμCa, would drive Ca2+ efflux, SV channel open probability was extremely low, and only at unphysiological conditions creating a ΔμCa driving Ca2+ into the vacuole, higher open probabilities were observed [2+], vacuolar [Ca2+], and membrane potential in a way to prevent vacuolar Ca2+ release. In the following years several studies presented evidence for cyt ≈ 2 μM, and one could question the physiological relevance of a vacuolar Ca2+ release brought about by an elevation of [Ca2+]cyt from 2 μM to 5 μM. For comparison, in animals cells the InsP3 receptor has maximum activity at [Ca2+]cyt ≈ 300 nM . It. ItTPC1  by Zn2+ . There aipsoidea  and Chloriabilis ) do not  crispus  and Pyroezoensis  seem to plastida , and it 2+ release [+ by ADP-ribosyl cyclases  increase in [Ca2+]cyt; this activates (nearby) InsP3 and ryanodine receptors, and Ca2+-induced Ca2+ release from the ER causes an effective (and widespread) [Ca2+]cyt elevation cyt changes induced by high external Ca2+ were identical in attpc1-2 and wild-type plants, indicating a role of AtTPC1 in stomatal closure in a signal pathway downstream of [Ca2+]cyt [2+]cyt increase, [Ca2+]cyt was recorded in attpc1-2 knockout, wild-type, and TPC1-overexpressing lines during the application of abiotic stresses  or biotic factors  [2+]cyt transients between lines lacking or overexpressing AtTPC1. Moreover, expression of stress-induced genes known to require [Ca2+]cyt increase did not show any dependence on TPC1 either. This seems to rule out an involvement of AtTPC1 in [Ca2+]cyt increase induced by the different tested stresses and elicitors cyt ,130. To e above) ,29.attpc1-2 has a wild-type phenotype, in the gain-of-function fou2 (fatty acid oxygenation upregulated 2) mutant older leaves have shorter petioles, show slight epinasty, and accumulate more anthocyanins, comparable to other mutants overaccumulating the plant hormone jasmonic acid  and lower vacuolar [K+] , which increases in Attpc1-2 mutants [2+]. However, whether this indicates that the SV/TPC1 channel functions as a vacuolar K+ efflux channel [2+ efflux channel [2+ sensor [While the loss-of-function mutant nic acid . In fou2+ and H+ . This islar [K+] , and discriptome . This hater CAX3 , which ificiency . However mutants . SV/TPC1 channel ,132, or  channel ,136, or + sensor ,136 seem2+ signaling, there are a few observations that still might support such a function. Application of high concentrations of sucrose induce a transient increase in [Ca2+]cyt in plant cells [2+]cyt increase was enhanced in plants overexpressing TPC1 and reduced in plant lines with suppressed TPC1 expression cyt induced by the fungal elicitor cryptogein was reduced in cell lines with suppressed NtTPC1 expression, as were defense-related gene expression and programmed cell death [Oryza sativa) a transient increase in [Ca2+]cyt induced by the fungal elicitor TvX was reduced in Ostpc1 knockout mutants cyt cyt . Cyclic 2+ is released from the large central vacuole by a Ca2+ channel belonging to the transient receptor potential (TRP) family  cyt transients in the cytosol. High activity of Ca2+ pumps, e.g., is expected to decrease amplitude and duration of [Ca2+]cyt transients  seem to contain more than a dozen Ca2+-ATPase genes, and plasma membrane and each organelle seem to contain at least one Ca2+-ATPase cyt transients were more than two-fold enhanced and dramatically prolonged. Moreover, knockout plants displayed reduced expression of stress-induced genes and decreased salt tolerance cyt transients were enhanced and programmed cell death was accelerated [2+-ATPases are shaping stimulus-induced [Ca2+]cyt transients and thereby may play a role in Ca2+-mediated signaling.Knockout mutants, lacking either ACA4 or ACA11, did not show obvious phenotypic differences compared to wild-type  lesions . Total lrecorded . The firolerance . Similarelerated . These s2+ pumps, it will always be difficult to differentiate what is a result of altered [Ca2+]cyt transients and what is a result of changes in ion homeostasis [2+] in a certain compartment may have unexpected results. Moreover, since Ca2+-ATPases transport both, Ca2+ and H+, pH homeostasis might be affected as well, as shown for Ca2+:H+ exchanger of the CAX family cyt transients were recorded. A. thaliana knockout lines lacking either AtCAX1 or AtCAX3 display an increased ABA sensitivity of seed germination cyt transients are still awaited.Most work on Caand Mn2+ ,176. Resf growth . In anotransport . Overexpignaling . Yet no mination , and themination ,176. For2+ signaling in plant cells (2+ channels. Available data provide evidence for InsP3 receptor and ryanodine receptor Ca2+ channels, the voltage-dependent SV/TPC1 channel, and most recently for vacuolar cyclic nucleotide-gated cation channels. The phylogenetic distribution of these Ca2+ channels among photosynthetic eukaryotes is patchy (3 or ryanodine receptors has been identified in land plants, after a very promising start in the 1990s, little progress has been made in our understanding of second messenger-mediated vacuolar Ca2+ release. As TPC1 was identified as encoding the SV channel, research on this field has seen a renaissance, and the SV/TPC1 channel is probably the best described ion channel in plant cells. However, we still do not understand the in vivo function of this channel; it is probably not Ca2+-induced vacuolar Ca2+ release. Progress in identification of genes encoding vacuolar Ca2+ pumps resulted in a better understanding of their physiological functions. In addition to maintaining Ca2+ gradients and ion homeostasis, P-type Ca2+-ATPases have been shown to affect the shape of [Ca2+]cyt transients, indicating that they might contribute more to Ca2+ signaling then just providing the driving force for vacuolar Ca2+ release.Most work on the involvement of the vacuole in Cant cells  has concs patchy  indicati


Mentoring is considered a valuable component of undergraduate medical education with a variety of programs at established medical schools. This study presents how new medical schools have set up mentoring programs as they have developed their curricula.Administrators from 14 US medical schools established since 2006 were surveyed regarding the structure and implementation of their mentoring programs.The majority of new medical schools had mentoring programs that varied in structure and implementation. Although the programs were viewed as valuable at each institution, challenges when creating and implementing mentoring programs in new medical schools included time constraints for faculty and students, and lack of financial and professional incentives for faculty.Similar to established medical schools, there was little uniformity among mentoring programs at new medical schools, likely reflecting differences in curriculum and program goals. Outcome measures are needed to determine whether a best practice for mentoring can be established. Mentoring programs in medical schools exist to provide students support and guidance that contribute to a fulfilling undergraduate medical experience , 2. AlthMentoring benefits may be seen in three major domains of the institution: the students, the mentors, and the medical school community. Potential benefits for students include career development, improved relationships with faculty, greater interest in research, aspirations toward academic careers, better academic performance, and emotional benefits, such as improved self-esteem and reduced stress , 3, 5, 6Although mentoring is undertaken for its benefits, mentoring programs face challenges as well. Mentors and mentees must have appropriate expectations of each other and mentors require the tools to effectively mentor an increasingly diverse student population , 3, 7. TFrei et al.  identifiAlthough the goals of most mentoring programs are similar, the methods and structure set up to achieve those goals differ greatly between medical schools. Mentoring programs range from structured with organized activities to unstructured with informal meetings between mentees and mentors –12, 14. The success of a mentoring program requires that it adapt to the changing needs of the medical school community. Meinel et al.  found thGiven the diversity of established medical school mentoring programs, the purpose of this study is to evaluate how mentoring programs are being established in new medical schools created since 2006. Specifically, we asked whether the variability in established programs is also found in new programs or whether areas of uniformity are emerging among schools starting mentoring programs at approximately the same time.www.lcme.org/directory.htm#pre-accredited-programs). All schools included were started in 2006 or later and had applied for Preliminary Accreditation from the LCME by August 2011. The inclusion criteria for schools to receive the survey were schools categorized into one of four categories: Applicant Schools, Candidate Schools, Preliminary Accreditations, and Provisional Accreditation.Participants were selected from the list of Developing Medical Education Programs obtained from the Liaison Committee on Medical Education (LCME) website  meeting of all new medical schools. If contact information was unavailable, then the medical school was excluded from the research study. In total, 14 medical schools participated , all in Given the focus of this survey on mentoring programs and the potential overlap between mentoring and advising, we initially set out to contrast the definitions of advising and mentoring for the instrument: ‘In a mentoring program, the relationship between mentor and mentee is reciprocal. The mentor listens and stimulates reflection in the mentee. In an advising program is the advisor is in control of the relationship. The advisor answers questions and gives advice, sharing their expertise and knowledge with the advisee’.Supplementary file).The initial questions for the mentor survey were constructed by the authors based on a review of the literature with an emphasis on the areas of mentoring programs outlined by Frei et al. . The queAn introductory email was sent in May 2012 to each student affairs’ contact, describing the research study and its purpose. Once consent was received, an email was sent containing a hypertext link to the online survey. Follow-up emails and phone calls were conducted every 2 weeks to non-responders to optimize participation. The Hofstra North Shore-LIJ School of Medicine Institutional Review Board approved the research study under exempt status.Quantitative data was analyzed using descriptive statistics in Excel.The researchers individually reviewed the qualitative survey data independently in order to conduct a preliminary analysis of the qualitative data. The individual members of the research team identified themes. The research team discussed the coded data and agreed on four themes: 1) the current state of mentoring in new medical schools, 2) steps in establishing a mentoring program, 3) benefits of the mentoring program to both mentors and students, and 4) challenges of the mentoring program to both mentors and students. This organization of the data under these themes supported the goal of the project. Then, the research team utilized these broad themes to do final coding of the survey data.All 14 eligible new US medical schools returned survey information but not all items were answered by every participant. Respondents were almost exclusively leadership from the Office of Student Affairs . Seven schools had preliminary accreditation, three had provisional accreditation, three were applicant schools, and one was a candidate school . All schAs described earlier, the survey introduction contained definitions contrasting mentoring and advising programs. We asked survey participants to direct their responses specifically to their mentoring programs. The majority of new medical schools in the survey population had both mentoring (79%) and advising (100%) programs, with combined programs reported by 42% of schools . The majNew medical schools reported the creation of mentoring programs was driven by two themes: 1) to provide students with role models and career guidance and 2) to create a ‘supportive atmosphere’ for students at their schools. One school responded the reason for establishing their mentoring program was ‘The need to provide students with the support of a mentor and a small group of students. The goals of our mentoring programs are: to provide a safe environment that encourages and fosters reflection, promote self-care and wellness, guide personal development, provide a resource for students seeking guidance, enhance team building and problem-solving skills, and assist in career exploration’. New mentoring programs emphasized student–faculty connections, professionalism, and career counseling, whereas less emphasis was given to wellness, stress reduction, and planning of student activities . SchoolsAlthough the surveyed schools had the same goals for implementing mentoring programs, the design of the programs to meet these goals was quite variable . For exaThe ratio of mentors to mentees varied greatly between programs ranging from 1:1 to 1:20 A. This rThe data reported here is the perspective of the institutional official completing the survey and may not reflect the views of individual faculty. Given the busy schedule of academic physicians, we were interested in learning how new medical schools recruit faculty to mentoring programs and how medical school faculty and administration view participation in mentoring programs. In general, faculty responded positively when asked to participate in mentoring programs. Survey respondents cited faculty time constraints as a primary reason given for not participating. Not surprisingly, the most common questions faculty asked when approached about participation in these mentoring programs involved time commitment and questions about specific activities. One school described the following set of questions by potential mentors ‘What is required to be a mentor? How much time will it take? How many meetings do I need to be involved in? Can I opt out of the program if I find it takes too much time or I don't think the arrangement with the student is working?’ Another school reported faculty had concerns about being unprepared to counsel students about personal issues.Six schools were not far enough into their mentoring program to determine the positive and negative effects of participating in a mentoring program for faculty and students. Among the eight respondents describing positive faculty aspects of mentoring, the most common comment listed by survey respondents was the opportunity to build stronger relationships with students. The second theme that emerged was an opportunity to stay current in academic medicine. One respondent stated mentoring ‘keeps you academically “on your toes,” an opportunity to share your wisdom and experience with a young promising doctor, rewards are priceless’. Positive aspects of mentoring programs reported by schools for students include learning from faculty who have ‘done it before’ and providing the students ‘professional and personal development’.Whereas time commitment was an issue for faculty, one school reported students were also having difficulty finding time to meet with their mentor. Interestingly, one school reported ‘some students feel that any activity that takes them away from studying is not worthwhile’. This feeling was identified by a second school as well.No mentors participated in the writing of the mentor in medical student performance evaluation (MSPE). Of the 12 schools who responded, all reported that the MSPE is written or overseen either by a Dean from the Office of Academic Affairs/Education or a Dean from the Office of Student Affairs.All schools surveyed, except one, reported there are plans to evaluate the newly established mentoring programs. Those responsible for evaluating mentoring programs differed between schools, with respondents listing assessment offices, course leadership, students, and the Office of Student Affairs. The common method of evaluation among all responding schools was sending surveys to students to determine their satisfaction with the mentoring program.Multiple studies report the value of mentoring programs in undergraduate and graduate medical education –3. Swan-Despite these common roles for mentors and common goals for mentoring programs, the structure of new mentoring programs to achieve these goals was highly variable in a number of important areas making a ‘best practices’ conclusion very difficult ; Fig. 1.Characteristics of failed mentor–mentee relationships include personality differences and poor communication, suggesting that student choice of the mentor might foster a more positive mentee experience than a randomly assigned mentor . HoweverTraining in mentor programs seems to be a priority for new schools and might be considered best practice as recent data demonstrates that graduates of a mentor training program utilize the learned skills in mentoring relationships . InteresAn important recent area of emphasis in undergraduate medical education is the creation of student wellness programs. Drolet and Rodgers, in their description of the Vanderbilt Wellness Program, describe three core areas: mentoring and advising, student leadership, and personal growth . Mentors‘Hidden curriculum’ is a term used to describe messages that are publicized as important but are not supported with a formal educational structures and resources . MentoriWe hypothesize that the formal structure, resource allocation, and most importantly the preservation of protected time for faculty are necessary for mentoring programs to flourish and have successful outcomes, both educational and professional. Additionally, whether the informal aspects of mentoring programs are recognized and supported with appropriate resources will likely have an impact on the success of the program, especially as class size increases and the new schools evolve and mature over time. This has the potential to limit the success of a well-intended and well-designed mentoring program. Frei concludes once mentoring program benefits are explicitly documented, including cost effectiveness, mentoring will receive more appreciation within institutions .A challenge to demonstrating the value of mentoring programs is the outcomes measured. The evaluation of mentoring programs in established programs and, according to our data, in new schools as well focuses on student and faculty satisfaction rather than tangible educational outcomes , 11, 20.Among millennial medical schools, the relationship between mentoring and advising programs is not always distinct and requires clarification. Limitations of this study include that half of the new schools reported a combined mentoring–advising program, suggesting in some cases the data may reflect an overlap between mentoring and advising. Despite distinct definitions in the literature that were included at the beginning of this study's survey instrument, the separation of these roles is not always distinct. The separation or combination of mentoring and advising roles has an impact on the planned responsibilities of the professionals who serve in these roles and may affect the quality of either program. An initial step for any school setting up a mentoring program is to clarify for students and faculty the roles and responsibilities aligned with mentoring and advising.Because of focus of this survey on new medical schools, existing surveys were not ideal. Therefore, our survey was self-designed by the research team based on published literature about mentoring programs. Additionally, data collections were limited to a single administrator view of the schools’ mentoring program. Views of faculty and students represent the perspective of the administration and were not directly reported by them. We have collected student data through a separate survey, and this data analysis is ongoing. Although we included open-ended survey questions, our data collection would benefit from structured interviews to obtain additional details.Previous surveys of the literature demonstrate that there is no single best practice for the design and execution of mentoring programs , 11, 12.


Drosophila is available, not much is known about their nature in other insect species. Here, we present description of some basic properties of both elementary and macroscopic LICs and their Ca2+-dependence in the photoreceptors of a dark-active species, the cockroach Periplaneta americana. Cockroach photoreceptors respond to single photon absorptions by generating quantum bumps with about 5-fold larger amplitudes than in Drosophila. At the macroscopic current level, cockroach photoreceptors responded to light with variable sensitivity and current waveform. This variability could be partially attributed to differences in whole-cell capacitance. Transient LICs, both elementary and macroscopic, showed only moderate dependence on extracellular Ca2+. However, with long light pulses, response inactivation was largely abolished and the overall size of LICs increased when extracellular Ca2+ was omitted. Finally, by determining relative ionic permeabilities from reversals of LICs, we demonstrate that when compared to Drosophila, cockroach light-gated channels are only moderately Ca2+-selective.In a microvillar photoreceptor, absorption of an incident photon initiates a phototransduction reaction that generates a depolarizing light-induced current (LIC) in the microvillus. Although in-depth knowledge about these processes in photoreceptors of the fruitfly  Limulus ventral eye , which, in turn, activates the effector enzyme phospholipase C (PLC). The active PLC then hydrolyses a membrane lipid component phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Transient receptor potential (TRP) and TRP-like (TRPL) channels are then opened apparently by both chemical   can be considered as a nocturnal species that shows clear visually guided behavior . Animals were received as adults from Blades Biological Ltd  and were subsequently kept at 25°C under a 12 h day/night rhythm.All experiments were performed on adult male cockroaches  and 0.2 mg/ml pancreatin (Sigma-Aldrich), retina pieces were gently triturated until ommatidia separated. Individual ommatidia, obtained after trituration with custom-made glass micropipettes, were allowed to settle in the recording chamber on the stage of an inverted microscope .Cockroaches were anesthetized for capture with COPatch-clamp recordings were performed as described before -methyl-2-amino-ethanesulfoncic acid (TES), 25 proline and 5 alanine, pH was adjusted to 7.15 (NaOH). Nominally Ca2+-free bath solution (“0” [Ca2+]) contained (in mM): 120 NaCl, 5 KCl, 4 MgCl2, 10 TES, 0.5ĖGTA, 25 proline, and 5 alanine, pH 7.15 (NaOH). Intracellular solution contained (in mM): 140 KCl, 10 TES, 2 MgCl2, 4 Mg-ATP, 0.4 Na-GTP, and 1 NAD, pH was adjusted to 7.15 (KOH). When 10 mM EGTA was used in the intracellular solution it was loaded with 4 mM CaCl2 to obtain ~150 nM and ~6 mM free Ca2+ and EGTA, respectively . The intracellular solution for recording of reversal potential (revE) under bi-onic conditions contained (in mM): 120 CsCl, 15 TEA-Cl, 10 TES, adjusted to pH 7.15 . The extracellular solution for recording of revE of monovalent ions contained (in mM): (1) 130 NaCl, 10 TES, 25 proline and 5 alanine, pH 7.15 (NMDG) for measurement of revE for Na+.; (2) 130 LiCl, 10 TES, 25 proline and 5 alanine, pH 7.15 (NMDG) for measurement of revE for Li+; and (3) 130 KCl, 10 TES, 25 proline and 5 alanine, pH 7.15 (NMDG) for measurement of revE for K+. Extracellular solutions for revE of divalent ions were (in mM): (1) 10 CaCl2, 120 NMDG-Cl, 10 TES, 25 proline and 5 alanine, pH 7.15 (NMDG) for measurement of revE for Ca2+; (2) 10 MgCl2, 120 NMDG-Cl, 10 TES, 25 proline and 5 alanine, pH 7.15 (NMDG) for measurement of revE for Mg2+; and (3) 10 BaCl2, 120 NMDG-Cl, 10 TES, 25 proline and 5 alanine, pH 7.15 (NMDG) for measurement of revE for Ba2+. The presence or absence of Cl− in the solutions did not change the properties of the LIC. All chemicals were purchased from Sigma Aldrich unless stated otherwise.Cockroach bath solution contained (in mM): 120 NaCl, 5 KCl, 4 MgClm is the mean, p is the fitted parameter, pkt is the time-to-peak, and s is the skewness.Quantum bump analysis was performed in MatLab , and was mostly based on the analysis by Henderson et al. . Bumps wnp for the occurrence of n responses per time period will follow the Poisson distribution:m is the mean number of responses, i.e., absorbed photons, which can be estimated from the probability of failed absorption (n = 0):If a light response consists of discrete responses, the probability n or more photons are required to elicit a light response follows the cumulative Poisson distribution:The probability that Q), follows the Gaussian distribution, the average size produced by a single photon absorption can be estimated via a sum of Gaussian functions weighted according to the Equation (3):0 is the standard deviation of failed responses and σ is the standard deviation of single photon response Q. μ is the mean Q of a single photon response, estimated from relation aQ/m, where aQ is the overall mean Q of failures and successful responses. The Poisson statistics presented in this study are based on PM:Pp < 0.05) were performed in Origin 9. Numerical results are presented either as mean ± standard deviation or median followed by interquartile range in brackets .Data analysis and statistics were performed with Origin 9  and MatLab . The comparison of Spearman's ρ correlation coefficients, sample normality tests  and the Mann-Whitney tests  many types of dynamics of the light-response , 50% rise time (T1), 50–100% rise time (T2), 50% decay time (T3), and peak amplitude (A)  Figure . The Q on = 1. To test this we made a series of repeated light flash experiments where the intensity of a light flash was varied by changing its duration . Each set of intensities (100–200 repetitions) produced a successful response at a different rate. These rates, or the frequencies of seeing, were then plotted against flash intensities  both A and T3 had a tendency to drift until reaching a stable level, while T1 and Q remained relatively stable throughout the recording period  was 58 (49–69) ms, and the average pkt and average half-width  in the five analyzed cells, the only correlations showing consistency were those between A and T3 (ρ = 0.17–0.47), and T2 and T3 (ρ = 0.26–0.46). These results suggest that at least latT and bump waveform (including A) are generated by processes independent of each other.Interdependence of bump generation processes can be assessed by examining correlation coefficients between bump parameters , the amplitudes narrowed down into a tighter group, which indicates that, except for the difference in sensitivity, the cells are functionally similar . In conclusion, since capacitance is an indirect measure of membrane area, the sensitivity differences may arise from differences in the size of rhabdomeres.As it was expected from earlier studies  and 1 ms flashes in one cell. The concentration series used consisted of 7 different solutions covering a range from 1.5 mM to nominally Ca2+-free . The concentrations were chosen following the study by Henderson et al.  indicated that in Ca2+ concentrations from 1.5 mM down to 25 μM the bumps became only slightly smaller and slower . The comparison of samples within each cell indicated a statistically significant difference only in one out of three cases. Another observation made in the same cells was that the latT dispersion appeared to increase when Ca2+ was omitted. The difference in median latT (cell 1: 43 (37–49) ms; cell 2: 45 (36–55) ms; and cell 3: 79 (63–96) ms under control conditions vs. cell 1: 53 (44–68) ms; cell 2: 59 (47–71) ms; and cell 3: 104 (89–162) ms in “0” [Ca2+]) was statistically significant within all three cells . In addition, the half-widths of lognormal fits of the latT distributions indicated that Ca2+ removal increased the dispersion of latT .To see how cockroach LIC is dependent on Can et al. . Surpris] Figure . With “0r Figure . Althougs Figure . Therefo2+ manipulations were then tested with macroscopic LICs. First, the same set of Ca2+ concentrations as above was used with responses to a 10 ms flash in one cell  until “0” [Ca2+] was used. In “0” [Ca2+], the LIC amplitude (from ~4 to ~10 nA) and duration increased dramatically. The second test involved using 10 s light pulses at different light levels before and after omission of extracellular Ca2+. The use of longer light pulses allowed us to observe changes beyond transient responses, and see how extracellular Ca2+ affects light adaptation. Since bump discrimination during continuous stimulation was practically impossible we had to rely on comparing effects using relative light intensities. The omission of extracellular Ca2+ resulted in an increase of amplitude and slightly prolonged duration of the LIC when compared to 1.5 mM Ca2+  was necessary for response excitation in the cockroach, EGTA should be able to inhibit the activation of the LIC. The effects of 10 mM EGTA were first tested on bumps. Figure A over a 870 s (14.5 min) light stimulation period started right after achieving the whole-cell configuration. Visual examination suggests that after 100 bumps A started to deteriorate. The frequency of the appearance of bumps also decreased with time, which can be seen from the sub-linear relation of bump number with time  ms. However, although showing otherwise similar behavior with EGTA, bumps recorded in another cell resulted in much shorter latT, with median of 38 (34–45) ms (N = 110), making definitive conclusions about the effects of EGTA on transduction impossible. We then tested how a prolonged exposure to intracellular EGTA would affect the waveform of the macroscopic response. This was done by first recording a LIC induced by a relatively bright 10 s light pulse right after formation of the whole-cell recording configuration. After 25 min:s the same light pulse was repeated. The resulting LIC showed a clear drop in transient amplitude, and was reminiscent of a response recorded with lower light intensity in control conditions. This behavior was in line with the drop in quantum efficiency observed with bumps recorded in the presence of EGTA. In the same cell, the effects of extracellular Ca2+ removal were also tested  mV  and 0.9 (0.9–0.9), respectively, giving a ~16:1 selectivity for Ca2+ over Na+.The surprisingly large size of cockroach quantum bumps in “0”  bath solution indicate that a significant fraction of the LIC could be mediated by an influx of Na+. It is consequently difficult to make solid conclusions about the sources of Ca2+ entering the cell. Nevertheless, an interesting finding was that in the brightest light level, the Drosophila trp phenotype could be reproduced with as well as without extracellular Ca2+ (Figure 2+ through capacitive Ca2+ entry (Stieve and Benner, Assuming that the influx of Ca Hardie, . Interes Figures . However Figures , suggest Hardie, . It is p+ Figure . It is t+ Figure  or the p2+ may not be such a predominant mediator of the LIC as it is in Drosophila photoreceptors (Figure 2+ was only ~16 times more permeable than Na+. For comparison, in Drosophila photoreceptors, Ca2+ is roughly 39 times more permeable than Na+ (Reuss et al., 2+ concentrations. The large size of Ca2+-free bumps also suggests that Na+ is responsible for a considerable fraction of the LIC. An intriguing possibility would be that cockroach photoreceptors also utilized TRP- and TRPL-type channels but with different proportions, favoring the amount of TRP less than in Drosophila. One of the remaining questions is also whether the Ca2+ influx via light-gated channels alone was enough to raise the intracellular Ca2+ levels sufficiently for proper regulation of transduction events.The results obtained with bumps led us to believe that Cas Figure . The perThe 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.


Background: Palmitoleic acid is a major pancreatitis-inducing agent.Results: Insulin protected cells from palmitoleic acid (POA)-induced ATP depletion, inhibition of the plasma membrane calcium pump (PMCA), cytotoxic Ca2+ overload and necrosis.Conclusion: Insulin protects against acinar cell injury induced by pancreatitis-inducing agents.Significance: This provides an important therapeutic strategy for treating pancreatitis with insulin therapy. 2+ ([Ca2+]i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca2+]i are linked critically by the ATP-driven plasma membrane Ca2+-ATPase (PMCA) important for maintaining low resting [Ca2+]i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca2+]i was measured by fura-2 imaging. An in situ [Ca2+]i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mm) and POAEE (100 μm) induced a small but irreversible Ca2+ overload response but had no significant effect on PMCA activity. POA (50–100 μm) induced a robust Ca2+ overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca2+ overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca2+ overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002. These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis.Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca Metabolr injury . TherefoOne way in which this might be achieved is by treatment with insulin. Evidence suggests that insulin may ameliorate the course of clinical acute pancreatitis –11 and p–12–2O2 mimic many of the cellular events during pancreatitis, this is not the most patho-physiologically relevant pancreatitis-inducing agent and might have additional effects not observed during pancreatitis. Therefore, the aim of the current study was to test the protective effects of insulin on pancreatic acinar injury induced by bona fide pancreatitis-inducing agents, such as ethanol and ethanol/fatty acid metabolites, including palmitoleic acid ethylesters (POEE) and palmitoleic acid (POA)  .2+ overload and necrotic cell death that was due to ATP depletion and inhibition of the PMCA. Consistent with our previous study, insulin pre-treatment either markedly attenuated or prevented all these POA-induced responses. Moreover, the protective effects of insulin were due in part to activation of PI3K/Akt. These data provide further evidence that insulin directly protects pancreatic acinar cells against bona fide pancreatitis-inducing agents.The results show that in isolated rat pancreatic acinar cells, POA was more cytotoxic than either POAEE or ethanol itself, consistent with previous studies –29. POA –w/v) bovine serum albumin  in a HEPES-buffered physiological saline solution . The inflated pancreas was then rapidly chopped with fine scissors and allowed to incubate for 30 min, replacing the solution after 15 min. Tissue clusters were then incubated with collagenase-free, Ca2+-free HEPES-PSS, containing 1 mm EDTA, for a further 5 min. This was followed by trituration to break up the tissue into small clusters, which were then filtered through a nylon mesh and centrifuged in 4% BSA HEPES-PSS (w/v) for 5 min at 300 × g. Following digestion, cells were resuspended in HEPES-PSS containing 0.1% BSA and kept on ice until use. Animals were humanely killed by an approved method as set out in Schedule 1 of the UK Animals Scientific Procedures Act 1986 (Certificate of Designation No 50/2506).Pancreatic acinar cells from Sprague-Dawley rats were isolated by collagenase digestion as previously described , 31. Brim fura-2-AM  for 30 min at room temperature in HEPES-PSS as previously described (2+]i using the well established method as previously described (42−-free HEPES-PSS to avoid precipitation with high concentrations of Ca2+. All experiments were carried out at room temperature (20–22 °C).Pancreatic acinar cells were loaded with 4 μescribed . For all2+-ATPase (SERCA) inhibitor, cyclopiazonic acid (CPA) in zero external Ca2+. This induces ER Ca2+ depletion and activation of store-operated Ca2+ entry (SOCE). Addition of 20 mm external Ca2+ to cells therefore results in a rapid increase in [Ca2+]I, which reaches a short-lived steady state. The subsequent removal of external Ca2+  causes a rapid [Ca2+]i clearance due predominantly to PMCA activity. Repeated Ca2+ influx-efflux phases allow test reagents to be applied during the second phase and compared with the first phase. [Ca2+]i clearance rate (and thus PMCA activity) was quantified by measuring the linear rate from a standardized value of [Ca2+]i during the second clearance phase and normalized to the corresponding linear rate  during the first clearance phase  to induce maximum ATP depletion. All responses were normalized to the maximum ATP depletion induced by the ATP depletion mixture  for a further 20 min before treatment with or without the ATP depletion mixture for a further 20 min. The total luminescence count of the ATP-depletion mixture was subtracted from each corresponding assay condition prior to normalization to the corresponding time-matched controls (%).ATP depletion was determined using two complementary techniques; magnesium green (MgGreen) and fire-fly luciferase  as previously described . Cells w mixture . For them and was allowed to incubate for 30 min prior to the acquisition of images. Corresponding brightfield and fluorescent images (3–5 images per well) were captured using a ×20 objective lens at 5 × 5 binning. PI-stained cells were excited with 545 ± 10 nm excitation light using a Cairn Monochromator  and emitted light was separated from excitation light using a TRITC dichroic . Fluorescent images were captured onto a CoolSNAP HQ CCD camera (Photometrics). Image acquisition was controlled by MetaFluor imaging software (Molecular Devices) and TIF images analyzed using Image J.Necrotic cell death was assessed using the cell impermeable nuclear-staining fluorescent dye, propidium iodide (PI). Pancreatic acinar cells were seeded into 96-well plates containing HEPES-PSS at an optimum density that was empirically determined to ensure maximum coverage of each well without compromising the ability to count the cells due to overcrowding. PI was added to each well from a ×100 stock to give a final concentration of 1.5 μm POA for 30, 60, 120, or 180 min prior to image acquisition. The minimum period of 30 min was chosen as this was the minimum period required for optimum PI staining; therefore POA and PI were added at the same time for the 30-min period, whereas PI was added 30 min before the end of each other subsequent period. For insulin-treated cells 100 nm insulin was added 20 min prior to the addition of either POA and/or PI. Mean (± S.E.) % PI-positive cell count (% dead cells) was determined from five separate cell preparations.In initial experiments total cell count was determined using the cell permeable nuclear staining fluorescent dyes, DAPI or Syto-16. This was done in an attempt to normalize PI-positive cell count (as a %) to the total cell count (either DAPI- or Syto-16-positive cells). However, it became apparent that both these dyes, routinely used to assess total cell count, did not always label every cell (either live or dead), which therefore under-estimated the total cell count and overestimated the % dead cell count. Therefore, total cell count was instead determined from brightfield images. Each field of view contained around 600–900 cells. Multiple fields of view –5 were t–2+]i were quantified using both the maximum change above resting [Ca2+]i and area under the curve (AUC) and responses compared using a one-way ANOVA with Bonferroni correction. For [Ca2+]i clearance experiments, a paired experimental design was adopted, in which two repeated Ca2+ clearance phases were elicited and the normalized linear [Ca2+]i clearance rate was compared with time matched control experiments using a Kruskal Wallis with post hoc Dunns test  and ethanol (100 mm) caused a small but irreversible increase in resting [Ca2+]i by 134 ± 9 and 162 ± 12 nm, respectively  and AUC of 94 ± 6 and 89 ± 6 μm.s (E). POA (100 μm), on the other hand, caused a much greater irreversible increase in [Ca2+]i by 487 ± 40 nm (C and D) and AUC to 247 ± 40 μm.s (C and E), consistent with previous studies in mouse pancreatic acinar cells , and palmitoleic acid (POA) , 25. To ± 6 μm.s E. POA .Because POA appeared to be the most cytotoxic pancreatitis-inducing agent tested, we next wanted to test whether insulin pre-incubation would prevent this POA-induced i by 224 ± 48 nm (n = 6), which was reduced to 63 ± 18 nm in the presence of insulin . Likewise, the POA-induced increase in AUC  was also markedly reduced in insulin-treated cells . Similarly the 100 μm POA-induced increase in [Ca2+]i was reduced from 407 ± 139 nm (n = 6) to 83 ± 26 nm  by insulin. Likewise, AUC  was also markedly reduced by insulin . Collectively, these data clearly show that insulin protects pancreatic acinar cells against POA-induced cytotoxic [Ca2+]i overload.On average, 50 μm) prior to treatment with 100 μm POA. Responses were then compared with parallel experiments performed on the same day in which cells were treated with either POA alone or POA with insulin pre-treatment (A and B). This concentration of POA (100 μm) was chosen because it produced the most robust Ca2+ overload response and was almost completely abolished by insulin pre-treatment  and insulin (100 nm) appeared to partially reverse the protective effect of insulin . Specifically, the maximum increase in POA-induced [Ca2+]i with insulin and LY294002  was completely reversed; there was no difference compared with POA alone  but was significantly different from POA and insulin . However, the AUC for the POA-induced [Ca2+]i with insulin and LY294002  was only partially reversed. The response was significantly lower than POA alone  and POA and insulin . This lower AUC was due to a significant delay in the rise in [Ca2+]i even though this eventually resulted in a similar maximal increase in [Ca2+]i. Nevertheless, these data suggest that activation of PI3K/Akt is only partially responsible for the protective effects of insulin and that another parallel signaling pathway may also be responsible.Insulin couples to numerous downstream signaling pathways including activation of intrinsic receptor tyrosine kinase activity, MAPK and PI3K/Akt activity. However, most of the acute short-term effects that do not involve changes in gene expression are mediated through PI3K/Akt. To test whether the protective effects of insulin were due to activation of PI3K/Akt pathways, cells were pre-treated with insulin in combination with the PI3K inhibitor, LY294002 . Initial experiments revealed that treatment with 50 μm POA during this second influx-clearance phase had no effect on clearance rate . This seemed counterintuitive, since 50 μm POA induced a robust irreversible [Ca2+]i overload (C and 2A). However, the time taken to reach the maximum increase in [Ca2+]i was between 10 and 20 min (C and 2A), whereas POA was applied on average 2 min prior to the influx-clearance phase . We therefore reasoned that POA must be applied for longer periods prior to measuring its effects on [Ca2+]i clearance. To test this it was necessary to carry out a series of time-matched control experiments in which various periods of time  were allowed between the recovery of the first clearance phase and the start of the second influx-clearance phase . However, after 20 min, [Ca2+]i clearance was significantly inhibited to 65 ± 6%, . Interestingly, however, in cells pre-treated with insulin this did not happen and [Ca2+]i clearance was maintained even after 20 min . This therefore suggests that insulin protects cells from the time-dependent inhibition of the PMCA.Using this approach, the second clearance rate was found to be on average 99 ± 2% of the initial clearance rate in time-matched control experiments n = 5;  and 5E. overload C and 2A.d 20 min C and 2A,ce phase . These e2+]i clearance was well maintained for up to 10 min between consecutive influx-clearance phases, the effect of POA treatments (50 and 100 μm) over these different time periods  was further tested with or without insulin treatment (100 nm for 30 min). As previously mentioned, over 2 min 50 μm POA had no significant effect on [Ca2+]i clearance either without  or with insulin treatment . However, 100 μm POA caused a significant inhibition to 59 ± 6% , which was completely prevented in insulin-treated cells . Over 5 min 50 μm POA now caused a significant inhibition of [Ca2+]i clearance to 64 ± 8% , which was completely prevented by insulin treatment . Similarly 100 μm POA caused an even greater inhibition of [Ca2+]i clearance over 5 min , which was also completely prevented in insulin-treated cells . Finally, over 10 min the inhibitory effects of POA were so severe , that insulin failed to exhibit any protection . However, these responses were highly variable, partly due to the fact that Ca2+ influx was also markedly inhibited, making analysis very difficult. Nevertheless, there was clearly a lack of protection by insulin under these conditions, suggesting that such a dramatic inhibition of Ca2+ clearance, regardless of any effect on Ca2+ influx, was so severe that insulin was unable to overcome this effect.Because [Ca2+]i overload. It is also worth noting that neither ethanol (as high as 100 mm) nor POAEE (as high as 100 μm), when applied for up to 10 min prior to the second influx-clearance phase had any effect on the Ca2+ clearance rate . However, following treatment with 100 nm insulin, the response to 100 μm POA was markedly reduced to 9 ± 2% ATP depletion .POA has been previously shown to induce mitochondrial depolarization, impair mitochondrial function and thus induce ATP depletion in pancreatic acinar cells , 32, 33.ith H2O2 . This waciferase , 30. MgGin vitro fire-fly luciferase chemiluminescence ATP assays, the experimental design was essentially the same as our previous study with H2O2 . The data were fitted to log transformed sigmoidal concentration-response curves which generated an average IC50 of 46 ± 1.1 μm and Hill slope of 2.1 ± 0.2 . Pre-treatment of cells with insulin (100 nm) caused a rightward shift in the concentration-response curve and significantly increased the average IC50 to 119 ± 1.1 μm . Surprisingly, insulin treatment alone (without POA) for the entire experimental period (3 × 20 min) significantly increased ATP levels compared with time-matched controls . Using both MgGreen and in vitro luciferase assays, these data suggest that insulin protects acinar cells from substantial ATP depletion induced by POA over the relatively short time period of 15–20 min.For experiments using the ith H2O2 . These etion see D. The dam POA significantly increased cell death to 64.4 ± 5.6% after only 60 min (p < 0.01), which continued to increase further at 120 min (79.6 ± 6.6%) and 180 min  dramatically and significantly attenuated the POA-induced cell death at 60 (22.7 ± 4.4%), 120 (36.3 ± 3.1%), and 180 min . PI is a cell impermeable nuclear-staining fluorescent dye that will only stain cell nuclei that have undergone cell lysis as a consequence of necrosis. Cell death was quantified by normalizing the PI-positive cell count to the total number of cells in the corresponding brightfield image (% cell death). In untreated time-matched control cells % necrotic cell death increased slightly from 10.4 ± 2.4% at 30 min to 21.1 ± 3.2% at 180 min . This reO-transferase , store depletion and the activation of store-operated Ca2+ entry. However, it is important to note that these POA-induced [Ca2+]i responses were nearly always irreversible and subsequent stimulation with CCK had no effect. Although we cannot rule out an effect on SERCA and SOCE as a source of the increase in [Ca2+]i, the irreversible nature of these responses suggests that PMCA activity is also most likely severely inhibited. In the context of pancreatic acinar cell injury inhibition of the PMCA is likely more important than inhibition of SERCA. This is because inhibition of the PMCA, regardless of whether the SERCA is inhibited, will always induce an irreversible Ca2+ overload in pancreatic acinar cells. This is because the ER has a finite capacity to store Ca2+ whereas the PMCA pumps Ca2+ into an effectively infinite extracellular compartment. Moreover, the PMCA is the only Ca2+ efflux pathway, due to the lack of NCX expression . Therefore, protection of the PMCA might be an important therapeutic strategy for preventing Ca2+ overload and the consequent acinar cell necrosis.The nature of the POA-induced increase in i data, POA-induced inhibition of the PMCA was almost entirely abolished by insulin pre-treatment. This was certainly true when POA was applied for up to 5 min prior to the second influx-clearance phase. However, over longer periods of POA treatment inhibition of the PMCA appeared to be so “severe” that pre-treatment with insulin failed to overcome this inibition. This temporal effect of both POA-induced inhibition of the PMCA and protection by insulin could be explained by the rate and extent of ATP depletion induced by POA that is sufficient to inhibit the PMCA. Using MgGreen to dynamically assess ATP depletion, it took up to 15 min for POA to reach the maximum MgGreen response, which was never as much as the ATP depletion induced by the mixture. In addition, using the luciferase assay to assess ATP depletion, although insulin shifted the concentration-ATP depletion response curve to the right, there were clearly higher concentrations of POA where insulin failed to have any protective effect. These data therefore suggest that insulin pre-treatment preserves cellular ATP to some extent during POA-induced cellular stress. Regardless of the absolute ATP concentration, this seems to be sufficient to maintain PMCA function and prevent the deleterious effects of cytotoxic Ca2+ overload.As alluded to above, ATP depletion is likely to affect both SERCA and PMCA; however, the irreversible nature of the POA-induced Cain situ [Ca2+]i clearance assays. This is not surprising as the cells are likely to be under considerable stress under the conditions of these experiments. Moreover, the PMCA is a major consumer of cellular ATP  time-matched control ular ATP , 52, theOur previous study showed that insulin switches metabolism from mitochondrial metabolism toward glycolysis . This ap45Ca2+ flux (and thus PMCA activity) into isolated inside-out plasma membrane vesicles from pig stomach smooth muscle cells showed that the PMCA has its own glycolytic ATP supply (i.e. >1 mm). However, in the face of impaired mitochondrial metabolism a “privileged” glycolytic ATP supply to the PMCA may become critical for maintaining low resting [Ca2+]i and thus avoiding stress-induced necrosis.Nevertheless, there are a few primary candidates. Phosphofructokinase-1 (PFK1) converts fructose-6-phosphate to fructose-1,6-bisphosphate and represents the major rate-limiting step in glycolysis. PFK-1 can be activated by insulin via tyrosine kinase-mediated phosphorylation . Phospho55–P supply , 61. ThiIn summary, the current study combined with our previous study provide compelling evidence that insulin has a direct protective effect on pancreatic acinar cells. This therefore suggests that insulin therapy with tight glycemic control, for example using the hyperinsulinaemic-euglycemic clamp, might be an effective treatment for severe acute pancreatitis. Moreover, the current study provides a strong rationale for further studies dissecting the molecular mechanism for the insulin-mediated protection of the PMCA in pancreatic acinar cells so that more specific drugs can be designed to treat pancreatitis without the deleterious effects of inadvertent hypoglycemia from insulin therapy.