Single channel recordings revealed reduced voltage/Ca2+ sensitivity of BKCa channels. BKCa α-subunit expression was unchanged, BKCa β1-subunit and sensitivity of BKCa to tamoxifen were reduced in the caffeine offspring as altered biophysical properties of BKCa in the MA. Simultaneous [Ca2+]i fluorescence and vasoconstriction testing showed reduced Ca2+, leading to diminished BKCa activation via ryanodine receptor Ca2+ release channels (RyRs), causing enhanced vascular tone. Reduced RyR1 was greater than that of RyR3. The results suggest that the altered STOCs activity in the caffeine offspring could attribute to down-regulation of RyRs-BKCa, providing new information for further understanding increased risks of hypertension in developmental origins.Caffeine modifies vascular/cardiac contractility. Embryonic exposure to caffeine altered cardiac functions in offspring. This study determined chronic influence of prenatal caffeine on vessel functions in offspring. Pregnant Sprague-Dawley rats (5-month-old) were exposed to high dose of caffeine, their offspring (5-month-old) were tested for vascular functions in mesenteric arteries (MA) and ion channel activities in smooth muscle cells. Prenatal exposure to caffeine increased pressor responses and vasoconstrictions to phenylephrine, accompanied by enhanced membrane depolarization. Large conductance Ca2 Activation of BKCa channel in the cell membrane allows K+ to flux out of the cell, causing hyper-polarization and consequent inhibition of voltage-dependent Ca2+ channels (VDCCs), resulting in vascular relaxationCa and VDCCs plays an important role in membrane potential regulation and vasomotion.A recent study demonstrated that malnutrition during pregnancy altered pressor responses and vascular activity associated with functional changes in Large conductance CaIt is rational that high concentrations of daily consumption of caffeine, instead of lower concentrations, are more likely to cause health concerns for under-matured fetuses. Thus, we designed a protocol of chronically using 20 mg/kg caffeine, twice a day, for a total caffeine of 40 mg/kg/day over almost whole pregnancy in rats. Based on the dose-conversion correlation between humans and rats (human: rats = 1:6.17), 40 mg/kg/day roughly equivalent to 4 cups of coffee (a cup of coffee contains nearly 150 mg of caffeine on average)In hypothesizing that the prenatal insult may affect vascular functions related to ion channel mechanisms, the present study was designed to investigate following questions: Did chronic prenatal exposure to high concentrations of caffeine increase risks in development of vascular diseases in life later? Whether and how ion channels were involved in prenatal caffeine mediated-impact on vascular dysfunction?Prenatal caffeine  significantly decreased fetal body weight to 86.73% compared to the control, with 25.1% of intrauterine growth restriction (IUGR) . HoweverG-nitro-L-arginine (L-NNA), the maximal response and pD2 values of PE-induced vasoconstrictions were significantly higher in the mesenteric arteries (MA) in the caffeine offspring (Table S2). In both the control and caffeine offspring, L-NNA significantly potentiated phenylephrine–induced vasoconstrictions without significant differences between the two groups (Em) for vascular re-activity in MA myocytes. As shown in Em was the same between the control (−45.67 ± 2.96 mV) and caffeine group (−44.67 ± 4.05 mV). Phenylephrine was more effective in depolarizing the myocytes in the caffeine offspring (Δ 35.67 ± 1.45 mV) than that in the control (Δ 28.04 ± 2.31 mV), suggesting that membrane depolarization might be involved in mechanisms for over re-activity of arterioles in the offspring exposed to prenatal caffeine.In absence of the NO synthase inhibitor No groups , suggest2+ channels play a central role in the regulation of vascular tone by membrane potential. L-type voltage-gated Ca2+ channels (LTCCs) appear to be dominant in most vascular muscle cellsEm in the caffeine offspring, we tested KCl-induced MA constrictions with or without LTCCs selective inhibitor, nifedipine (10−9 ~ 10−5 mol/L). As shown in 1C protein in the MA of the caffeine group , a selective BKCa inhibitor, to determine the BKCa channel function in buffering phenylephrine-induced vessel contraction. Ca channel inhibition on phenylephrine-induced vessel contraction. After BKCa channel blocked by IbTX (10−8 M), the phenylephrine-induced vessel contraction was significantly enhanced ~66.8% (control: from 253.98 ± 29.14 to 422.92 ± 22.98%Kmax), whereas no significant increase can be observed in caffeine offspring  from the caffeine offspring.As shown in 2+ (Ca2+ sparks), caused by Ca2+ release from sarcoplasmic reticulum, are thought to trigger opening of BKCa channels resulting in STOCs in SMCsCa channels activated by a Ca2+ spark. As shown in Figure S2A, single BKCa channel conductance was the same between the two groups (250.27 ± 10.90 vs. 251.93 ± 13.56 pS). The α-subunit selective BKCa opener, NS-1619 (2 × 10−5 mol/L) exerted a similar effect on BKCa currents in the control and caffeine myocytes . Moreover, western blot analysis failed to reveal differences in the expression of α-subunits between the control and caffeine group .Localized and transient elevations in cytosolic Cane group . Because2+ release that activated a transient BKCa currents. Thus, we tested Ca2+/voltage sensitivity of BKCa channels. 2+]i. Both groups showed an enhanced NPo of BKCa channels with increasing Ca2+ concentrations, while the Po-calcium curve for the caffeine group was right shifted with an increased Kd value (control vs. caffeine: 0.92 ± 0.06 vs. 3.85 ± 0.06). There was no significant difference of the Hill coefficient between both groups (1.05 ± 0.03 vs. 1.08 ± 0.04). Po-voltage relations fitted by Boltzmann equation also were determined at 10−6 mol/L [Ca2+]i  was 47.29 ± 2.35 mV (Control) vs. 57.08 ± 1.79 mV (Caffeine). Those data showed both the calcium and voltage sensitivity of BKCa channels were attenuated in the caffeine MA.Another possible explanation for the reduced STOCs was a decrease in the percentage of sarcoplasmic reticulum (SR) Ca [Ca2+]i . There w2+/voltage sensitivity and kinetic stability of BKCa channels is known to be dependent on presence of β1 accessory subunitsCa channels when they were associated with β1 subunits−6 mol/L tamoxifen (10−7 mol/L [Ca2+]i) evoked approximately 7.31-fold of increase in the Po of BKCa in the control, while only 2.21-fold in the caffeine group  than those from the control (τopen = 10.26 ± 3.24 ms).Cane group . The expnchanged , showingfunction . BKCa ch2+ increase/Ca2+ sparks via ryanodine receptor Ca2+ release channels (RyRs) activates BKCa channels in generating STOCs and modulating vascular toneCa channels, the attenuated STOCs activity observed in the caffeine group could be resulted from the decreased Ca2+ release from the ryanodine sensitive stores.It is well established that transient Ca2+ release by opening of RyRs in the SR, which increases subsarcolemmal Ca2+ concentrations2+ responses to K+. 2+ responses and the corresponding arterial diameter in the same pressurized MA. The fluorescence ratio (F340/F380), indicating cytosolic Ca2+ levels−5 mol/L) in the same arterial segment, the KCl-induced increase in fluorescence ratio was significantly reduced by 33.14% in the control, where the reduction was significantly less in the caffeine group (16.70%), indicating that the Ca2+ release by activation of RyRs could be reduced in the caffeine group  BKCa channel function in buffering phenylephrine-induced vascular constriction was impaired by prenatal exposure to caffeine. Perforated patch recording demonstrated a reduced STOCs frequency/amplitude in the caffeine offspring. Single channel recordings revealed degraded biophysical/intrinsic properties of BKCa channels, including reduced Ca2+/voltage sensitivity and stability of open states. Ca2+ fluorescence imaging showed a reduced Ca2+ release from SR. Molecular biology approaches revealed a decreased expression of BKCa β1-subunit, RyR1, and RyR3 in the caffeine group.The main findings of the present study include: 1) Prenatal chronic exposure to high dose of caffeine significantly increased phenylephrine-stimulated pressor responses, vascular-activity, and membrane depolarization in the adult offspring; 2) Prenatal caffeine influenced LTCCs functions associated with reduced αPrenatal caffeine induced 25.1% IUGR in our experiments. IUGR is widely accepted as a critical cause for increased susceptibility of cardiovascular diseases2931Our initial ideas proposed that both vascular functions in vasorelaxation and vasoconstriction may be counted for the altered vascular responses to phenylephrine in the caffeine group. However, the data of inhibition of eNOS with L-NNA did not support that NO-dependent vasorelaxation may be involved. Thus, we focused on the increased vessel activity. In the present study, prenatal caffeine significantly increased pressor responses to the classic vasoconstrictor, suggesting an enhanced risk in development of hypertension in life later. Previous studies demonstrated that nicotine exposure in pregnancy increased angiotensin II-stimulated pressor responses in the young offspring2+ channels, e.g, an up-regulation of LTCCs in the genesis of hypertension and hypercholesterolemia3638Ca channel functions should be taken into consideration. Our investigation at cell level showed that BKCa currents/STOCs in the caffeine offspring were markedly reduced. Because STOCs act in hyper-polarizing and relaxing vascular SMCsCa channel Ca2+/voltage sensitivity and modulatory β1-subunits should be causes of the reduced STOCs. The BKCa β1-subunits modify functional properties of pore-forming α-subunits, including increasing channel Ca2+ sensitivity, slowing down macroscopic activation, and deactivating kinetics, and decreasing voltage sensitivityTo determine mechanisms underlying the increased pressor and vascular tone, we focused on resistance arteries and ion channels on smooth muscle cells. Numerous studies showed physiopathologic basis of vascular disorders involving dysfunction of L-type CaRYR1, RYR2, and RYR3)2+ release during Ca2+ sparks and global Ca2+ responses in vascular myocytes, evoked by activation of voltage-gated Ca2+ channels. RyR1 and RyR2 appeared functionally capable to couple with BKCa channels. In contrast to RyR1 and RyR2, Ca2+ spark triggering arterial tone is precisely adjusted by RyR3. RyR3 is part of the SR Ca2+ spark release unit, and feeds back RyR1 and RyR2 to decrease Ca2+ spark frequency. RyR3 can be activated by caffeine and local increases in [Ca2+]i under conditions of increased SR Ca2+ loading25432+ release from the SR. Although the decreased RyR3 may augment the Ca2+ spark and STOCs, the reduced STOCs activity and global Ca2+ responses by depolarizing (60 mM KCl) and caffeine could be resulted from stronger suppression of RyR1 than that of RyR3. To the best of our knowledge, this was the first to show altered RyRs by prenatal insults in exploring cellular mechanisms of vascular diseases in developmental origins.The mammalian RyRs are encoded by 3 different genes  auxiliary subunits45/− mice demonstrated that vascular SMCs expressed fewer LTCCs472+ influx mode” in which whole-cell LTCCs currents and α1c -subunit expression were decreased while local Ca2+ influx activity increased, resulting in an increased frequency and amplitude of RyRs-mediated Ca2+ sparks, acting as a feedback mechanism against the degraded coupling between sparks and STOCs. The molecular mechanism underlying high activity of LTCCs showed an increase in α1/α2δ/β2 channel-forming complexes, rather than α1/α2δ/β3, in myocytesL−Ca cross linking of sparks and cell-wide [Ca2+]i changes to altered membrane potentials deserves further investigation.We initially anticipated that an enhanced depolarization of myocytes following prenatal caffeine would be accounted, at least partially, by the increased LTCCs function. However, that was not the case. The functional roles of LTCCs were not enhanced but rather diminished according to the decreased channel currents and pore-forming αEm (~−40 mV)   was weakCa β1 subunits, insensitivity of BKCa to SR Ca2+ release, and increase of Em. The reduced RyR1, RyR3, and Ca2+ release via RyRs influenced BKCa activation/STOCs, leading to a poor negative feedback of Em in limiting vasoconstrictions. On the other hand, the expression of LTCCs was decreased, which probably occurred as a protective mechanism against the increased membrane potential and vascular re-activity. The present work focused on male offpsring only, future projects should consider the female too. Based on previous studies on both male and female offspring, there could be similar and different effects of prenatal insults on offspring between the male and female.Based on those findings, we proposed a model for a mechanistic explanation of enhanced myocyte depolarization and vasoconstrictions observed in the caffeine offspring . Chronic high dose of caffeine in pregnancy may cause a suppressed expression of BKCa signaling, which could be counted for the increased pressor responses as risk of hypertension in fetal origins. Significance of the findings includes that daily high doses of caffeine or probably too much high concentrated coffee or tea during pregnancy may be risk in disease development in fetal origins.In summary, the present study on resistance arteries was the first to raise the possibility that there is a selective down-regulation of β1 subunits accompanied by nonparallel suppression of RyRs resulting in an impairment of RyRs-BKSprague-Dawley rats were used and the experimental procedures were approved by the Institutional Committee of Soochow University and in accordance with the Guide for the Care and Use of Laboratory Animals .Male adult offspring (5-month-old) were implanted with catheters for recording of BP as describedmax).Small segments of mesenteric arteries (A3~A4) were isolated as described previouslyCa single channel currents were recorded from inside-out patches. Whole-cell K+ currents were measured in conventional whole-cell patch-clamp configuration. SMCs membrane potential was evaluated as previously describedSMCs from small mesenteric arteries were isolated51Small mesenteric arteries were collected and protein was extracted, estimated, and transferred to the membrane using a routine method.2+]i in mesenteric arteries was monitored using Ca2+ indicator Fura 2-AM , as described previously545556The arterial segments were mounted and pressurized in a chamber . [CaIsolated SMCs were loaded with Fluo-3 AM. For fluorescence imaging, the cell chambers were positioned on the stage of an Olympus IX81 inverted microscope equipped with a Xenon MT-ARC/XE system and an OBS NN10 CCD camera . Image acquisition and analysis was performed using xcellence rt01 .Total RNA was extracted and quantified. RNA samples were reverse-transcribed into cDNA using a routine method.Mesenteric arteries were homogenized with 20% ethanol in phosphate buffer solution (PBS), then centrifuged at 3000 × g at 4 °C for 5 min. Supernatants were collected for analysis using an ELISA kit  following the manufacturer’s protocol. Please see supplement online for details.t test or ANOVA, when appropriate, was used to determine significance of differences among groups. A probability value of <0.05 was considered statistically significant.Data are presented as mean ± SEM. How to cite this article: Li, N. et al. Chronic fetal exposure to caffeine altered resistance vessel functions via RyRs-BKCa down-regulation in rat offspring. Sci. Rep.5, 13225; doi: 10.1038/srep13225 (2015).


Based on the aequorin system, we describe the establishment of a calcium reporter line in rice and test the responses of the reporter towards salt and oxidative stress. 2+ ([Ca2+]i), which is caused by the flux of calcium (Ca2+). However, the differences between these two processes are largely unknown. Here, we introduced recombinant aequorin into rice (Oryza sativa) and examined the change in [Ca2+]i in response to salt and ROS stresses. The transgenic rice harbouring aequorin showed strong luminescence in roots when treated with exogenous Ca2+. Considering the histological differences in roots between rice and Arabidopsis, we reappraised the discharging solution, and suggested that the percentage of ethanol should be 25%. Different concentrations of NaCl induced immediate [Ca2+]i spikes with the same durations and phases. In contrast, H2O2 induced delayed [Ca2+]i spikes with different peaks according to the concentrations of H2O2. According to the Ca2+ inhibitor research, we also showed that the sources of Ca2+ induced by NaCl and H2O2 are different. Furthermore, we evaluated the contribution of [Ca2+]i responses in the NaCl- and H2O2-induced gene expressions respectively, and present a Ca2+- and H2O2-mediated molecular signalling model for the initial response to NaCl in rice.It is well established that both salt and reactive oxygen species (ROS) stresses are able to increase the concentration of cytosolic free Ca Oryza sativa L.) is the staple food for more than half of the world’s population. Salinity is one of the most common abiotic stresses encountered by rice, which is classified as a salt-sensitive crop in early stages of development, and limits its productivity (Rice (2+) spike, which is caused by the flux of Ca2+. This salt stress triggered increase of cytosolic free Ca2+ ([Ca2+]i) is considered to be the first recorded response to salt stress  signalling pathway, which comprises SOS3, SOS2 and SOS1 , and is  cascade .2O2  is required for shoot sodium homeostasis during salt stress  is reported to function as a central hub to regulate ROS-dependent signalling during the initial response to salt stress in rice  predominantly represented by Her  seeds were sterilized with 75% ethanol and planted in a square plate containing half-strength Murashige and Skoog salts , and 1.5% (w/v) agar (Becton Dickinson). Seedlings were grown vertically in the growth chamber conditioned with 16h of light at 28°C and 8h of dark at 22°C for five days. The seedlings were then sprayed with coelenterazine for reconstitution of aequorin before subsequent experiments began.Rice  and hybridized with a 32P-dCTP-labelled hygromycin-resistant gene probe. The blots were washed at 65°C under stringent conditions and analysed using Typhoon-8600. The primers used to amplify the probe are listed in Supplementary Table S1.Genomic DNA of transgenic rice was isolated following the instructions of a Plant Genomic DNA Kit (TIANGEN) and the purified DNA was digested with restriction enzyme OsACTIN. For the examination of NaCl and H2O2 induced gene expression, the roots of five-day-old rice seedlings were selected. Ca2+ channel blocker pre-treatment was performed by 1mM LaCl3 treatment for 30min. NaCl treatment was performed by 0.15M NaCl treatment for 1h. H2O2 treatment was performed by 1mM H2O2 treatment for 1h. The relative expression levels were calculated according to the 2-∆∆Ct method (12–18 (500 μg ml-1) primer, and 1μl of 10mM dNTP mixture . After addition of 4 μl 5× reaction buffer (Promega), the reaction was incubated at 37°C for 2min, and 1 μl (200 units) of M-MLV RTa (Promega) was added to the reaction and incubated at 42°C for another 50min. For inactivation, the reaction was heated at 70°C for 15min. The primers are listed in Supplementary Table S1.For the examination of apoaequorin expression in different tissues, the shoot, shoot base and root of five-day-old transgenic rice seedlings were selected, and the PCR was conducted with 28 cycles for both apoaequorin and t method . Each exin vivo by spraying seedlings with 10 µM coelenterazine and followed by incubation at 21°C in the dark for 12–16h. For surfactant treatment, 0.01% or 0.1% of silwet L-77 (Sigma) was added to the coelenterazine solution. For Ca2+ inhibitor treatments, rice roots were treated with different concentrations of GdCl3, LaCl3, neomycin and thapsigargin, respectively for 30min before 0.25M NaCl and 1mM H2O2 treatment. Treatments and aequorin luminescence imaging were performed at room temperature using a ChemiPro HT system as described previously  35S promoter . Ca2+-treated seedlings showed strong and diverse luminescence in roots, and AQ-3 with the strongest luminescence was selected for further analysis . It is likely that the leaf wax prevents the permeating of coelenterazine . To test this hypothesis, we added surfactant  while spraying coelenterazine. Both luminescence signals in roots and dotted signals in shoots were observed . These results showed that transgenic rice expressing apoaequorin was able to reflect the [Ca2+]i level in rice roots.In order to monitor i responses were made. As shown in 2+]i within 30 s, which quickly declined to the basal level after the spike. The amplitudes of luminescence signals varied according to different concentrations of NaCl, while both the durations and phases were the same. Detailed analysis showed that 0.1M NaCl had little effect on the induction of [Ca2+]i, while 0.15M NaCl was able to induce an obvious spike of [Ca2+]i. The effect of 0.2M NaCl was not significantly different in the increase of amplitude of [Ca2+]i increase compared with 0.15M. Interestingly, increasing the concentration of NaCl to 0.25M dramatically increased the amplitude of [Ca2+]i by about two fold, but concentrations higher than 0.25M NaCl had little effect on the increasing of amplitude compared with 0.25M .To investigate the time courses of [Cath 0.25M . These rreatment . This in2+]i changes in response to ROS stress in rice roots, we examined the aequorin-based luminescence after the application of H2O2. We collected the luminescence signals 45 times at one minute intervals after treatment with H2O2. We found clear luminescence signals in the first and second minute. Interestingly, after the weak luminescence signals in the third minute, we failed to collect any additional signals over the remaining 42min . This was different from that in Arabidopsis, which was reported to have a second peak 5–20min after the application of H2O2 . Higher concentrations of H2O2 (more than 0.5mM) were able to induce stronger luminescence signals but with a reduced rate of increase .To investigate the Hnescence . A concewould be . Detaileincrease . Concent2+]i induced by different concentrations of H2O2 in rice roots. H2O2 did not induce an immediate spike of [Ca2+]i as NaCl did. Our results showed that within the first 30 s, only the highest concentration of H2O2 (5mM) was able to induce any luminescence signal, this is in contrast to NaCl which within 30 s was able to induce luminescence signals for all but the lowest concentration (0.1M)  had a peak between 30 s and 1min, lower concentrations of H2O2 (below than 0.4mM) would delay the peak by about 30 s. Moreover, the spike of [Ca2+]i induced by H2O2 did not decline to the basal level as quickly as NaCl. The luminescence signals declined gradually to the basal level within 3min  . Furtherhin 3min . These r2+ in NaCl- and H2O2-induced [Ca2+]i responses, we tested GdCl3-, LaCl3-, neomycin- and thapsigargin-treated plants on the [Ca2+]i increase in response to NaCl and H2O2 respectively. Gd3+ and La3+ are agonists of Ca2+, and they have been used as Ca2+ channel blockers to inhibit Ca2+ flux , DEHYDRATION-RESPONSIVE ELEMENT BINDING2A (DREB2A) and STRESS-RESPONSIVE NAC1 (SNAC1), which were reported to be induced by NaCl and H2O2 in rice, were analysed by quantitative RT-PCR (qRT-PCR) . The luminescence signals in shoots were dotted, indicating insufficient permeation of coelenterazine. Although a higher concentration of surfactant (0.1%) increased the dotted luminescence signals in shoots, it greatly decreased the luminescence signals in roots, indicating the toxic effect of the surfactant to rice roots . These results showed that our system is able to reflect the [Ca2+]i level only in roots, not shoots.Since the transformation of aequorin in plants , and the central cylinder are present in a rice root, compared with single cell layers of epidermis, cortex, endodermis and the central cylinder in Arabidopsis  did not induce an obvious increase of [Ca2+]i, although it is sufficient to induce the expression of many salt response genes . Considering the different environments rice and Arabidopsis roots are adapted to, rice may have evolved a different mechanism to cope with ROS stress.We investigated the i changes in response to different concentrations of NaCl and H2O2 treatments in rice roots. Treatments of NaCl at different concentrations induced immediate [Ca2+]i spikes within 30 s , such as MAPK5. In our experiment, H2O2 did not induce the expression of DREB2A significantly as previously described (2O2 treatment may not be long enough to induce the expression of DREB2A. In the previous report, 3h of H2O2 treatment was able to induce the expression of DREB2A, while 30min of H2O2 treatment had no obvious effect (DREB2A (DREB2A (Recently, the molecular processes controlling early stress perception and signalling have been explored and several genes have been identified as initial response genes in rice (of LaCl3 . In the of LaCl3 . Conside in rice . Salt st (DREB2A , a compl2+]i responses to NaCl and H2O2 in rice roots. Ca2+ inhibitor treatments revealed that both external and internal Ca2+ stores are involved in the [Ca2+]i increases induced by NaCl and H2O2. Further research suggested that the internal Ca2+ store in ER participates in the NaCl-induced [Ca2+]i increase, while another internal Ca2+ store, rather than ER may participate in the H2O2- induced [Ca2+]i increase. In addition, we dissected the early salt-stress signalling cascade and presented a Ca2+- and H2O2-mediated molecular signalling model for the initial response to NaCl in rice.In summary, based on the aequorin luminescence, we established a calcium reporter line in rice. Using this, we tested the different [CaJXB online.Supplementary data can be found at Supplementary Fig. S1. Leaf wax prevents the permeating of coelenterazine.Supplementary Fig. S2. The optimization of discharging solution for luminescence imaging in rice.Supplementary Fig. S3. Treatment with high concentration of NaCl resulted in serious cell death in the rice roots.Supplementary Figure S4 H2O2 does not induce a second peak of [Ca2+]i response.Supplementary Fig. S4. H2O2 does not induce a second peak of [Ca2+]i response.Supplementary Fig. S5. High concentrations of H2O2 have little additional effect on the luminescence changes.Supplementary Table S1. The sequences of primers used in this research.


The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs  have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store.Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca Our results show that despite an ER targeting and ER retention sequences, cameleons containing cpEGFP on the C-terminus show poor ER localization. Moreover, the GFP/OFP-based ER targeted cameleons gained Ca2+ sensitivity, which make these probes impractical for sensing ER Ca2+ signals. Finally, we found suitable GFP/RFP FRET pairs for the development of functional ER targeted cameleons, which are suitable for dual recordings of [Ca2+]ER and [Ca2+]Cyto in single individual cells.In this study, we engineered numerous red-shifted ER-targeted cameleons based on the optimized D1 motif. Such bathochromic probes can be better used for simultaneous measurements of Cyto and [Ca2+]ER cells expressing one of the red-shifted cameleons were loaded with 3 µM fura-2/AM at room temperature for 30–40 min in a buffer composed of (in mM): 2 CaCl2 135 NaCl, 5 KCl, 1 MgCl2, 1 HEPES, 2.6 NaHCO3, 0.44 KH2PO4, 0.34 Na2HPO4, 10 D-glucose, 0.1% vitamins, 0.2% essential amino acids and 1% penicillin/streptomycin pH 7.4. Fura-2 loaded cells as wells as non-loaded cells were washed and stored in this buffer prior to imaging experiments. For Ca2+ measurements, cells were perfused in a HEPES buffered solution containing (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 1 HEPES and 10 D-Glucose pH 7.4 (0 mM Ca2+). Additionally, either 1 mM EGTA (EGTA), 1 mM CaCl2 (1 mM Ca2+) or 2 mM CaCl2 (2 mM Ca2+) were added to 0 mM Ca2+ and pH adjusted to 7.4.For dual recordings of  from D1ERCmR2 was determined in situ. The ER of permeabilized HeLa cells was depleted using a 3 mM EGTA buffer. Free Ca2+ concentrations were calculated using the CaBuff software  and appropriate amount of Ca2+ were added to the 3 mM EGTA buffer to obtain buffer solutions of 1, 3, 10, 30, 100, 300, 1000, 2000 and 10,000 µM free Ca2+.HeLa cells transfected with ER targeted cameleons were permeabilized with 5 µM digitonin and 10 µM ionomycin (digitonin/ionomycin) in 2 mM Caescribed  Signal t2+ imaging was done using the TiLL iMIC  digital wide field imaging system, as described previously ER.Analogous to recently developed red-shifted cameleons for imaging Ca1GO-Cam)  we constRGO-Cam2 A. For ERRGO-Cam2 B. ColocaRGO-Cam2 A consideRGO-Cam2 B. D1ERGOn the ER C. The po to 1 µM  Accordin2+ sensitivity). In total, we generated and tested 10 different ER targeted GFP/RFP-based cameleons that differ either in the FP-FRET pair or the orientation of FPs within the construct  µM for D1ERCmR2 , which indicates that the red-shifted cameleon allows better detecting changes of ER Ca2+ over a broad range.In order to verify whether or not GFP/RFP-based ER targeted cameleons have a D1ERCmR2 . Originaat 60 µM ,34. Howee 220 µM  which is2+ sensors allows simultaneous recordings of [Ca2+]ER and [Ca2+]Cyto in single individual cells. As expected, imaging of fura-2 loaded HeLa cells that express D1ERCmR2 revealed no fluorescence overlap of the respective fluorescence signals , the combination of both Ca signals A. Cell t-2 ratio , indicatost 10 s C. Moreovrestored C,D. Whil2+]Cyto and [Ca2+]ER was further tested using HEK-293 cells stably expressing the ryanodine receptor 2 (RYR2). RYR2 is referred to as cardiac Ca2+ release channel that makes HEK-293 cells (HEK E2) inducible to extracellular Ca2+ [2+ addition is known to activate store overload induced Ca2+ release (SOICR) in the HEK E2 clone. SOICR is frequently used to characterize the RYR2 channel activity using these heterologous expression system [2+]ER and fura-2 as an indicator for [Ca2+]Cyto, we observed cytosolic and ER Ca2+ oscillations in the HEK E2 clone (2+-dependent activation of the RYR2 in more detail. Particularly, the simultaneous recording of [Ca2+]Cyto and [Ca2+]ER in this cell model should allow determining the ratio of [Ca2+]Cyto/[Ca2+]ER upon clusters of RyR2 get activated. However, fura-2 as a Ca2+ buffer might affect the frequency of SOICR under these conditions.The approach of dual recordings of ER and [Ca2+]Cyto in single individual cells. With this work, we expanded the palette of red-shifted organelle targeted Ca2+ probes that show less phototoxicity and should be compatible with most of the existing confocal and wide-field fluorescence imaging systems.In this study, we show that GFP/OFP-based cameleons are impractical to monitor ER Ca


Exogenous application of caffeine, a ryanodine receptor agonist, induced oscillatory intracellular free Ca2+ concentration ([Ca2+]i) responses in a duration- and concentration-dependent manner. In Ca2+-free Ringer’s solution, [Ca2+]i transients could also be induced by a brief caffeine application, whereas subsequent caffeine application induced no [Ca2+]i increase, which implied that extracellular Ca2+ was required for ER refilling, confirming the necessity of a Ca2+ influx pathway for ER refilling. Depletion of ER Ca2+ by thapsigargin triggered a Ca2+ influx which could be blocked by the store-operated channel inhibitor 2-APB, which proved the existence of the store-operated Ca2+ entry pathway. Taken together, these results suggested that after being depleted by caffeine, the ER was replenished by Ca2+ influx via store-operated channels. These results reveal the fine modulation of ER Ca2+ signaling, and the activation of the store-operated Ca2+ entry pathway guarantees the replenishment of the ER so that the cell can be ready for response to the subsequent stimulus.The mechanisms of release, depletion, and refilling of endoplasmic reticulum (ER) Ca At the meantime, Ca2+ release from the ER is mainly mediated by two families of Ca2+ release channels, i.e., the inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR) families. While IP3Rs are gated by IP3, both IP3Rs and RyRs can be activated by Ca2+, and such Ca2+-induced Ca2+ release (CICR) forms a positive feedback process. RyRs can also be activated by caffeine, which sensitizes RyR’s response to Ca2+As the primary intracellular reservoir of Ca2+ channel currents 2+ dynamics of H1 HCs is still limited.Type I horizontal cells (H1 HCs) are interneurons in the outer retina of carp, which receive glutamate input from cone photoreceptors. It was found in the fish retina that HCs contained caffeine-sensitive ER 2+ dynamics of carp H1 HCs, particularly the details of the following aspects: (1) the temporal characteristics of [Ca2+]i signals initiated by ER Ca2+ release, (2) the inter-relationship between the two major Ca2+ sources, i.e., the extracellular Ca2+ and the ER, and relevant channels underlying their interaction.The present study aims to investigate possible mechanisms of ER Ca2+ signals elicited by exogenously applied caffeine were recorded from freshly dissociated H1 HCs using a fluo-3-based Ca2+ imaging technique. The basic findings are: (1) caffeine induced oscillatory [Ca2+]i responses in a duration- and concentration-dependent manner, (2) removal of extracellular Ca2+ abolished the repeatability of caffeine-induced [Ca2+]i responses, (3) inhibition of L-type voltage-gated Ca2+ channels (L-VGCCs) reduced caffeine-induced [Ca2+]i oscillations, (4) inhibition of store-operated channels (SOCs) abolished caffeine-induced [Ca2+]i oscillations. These results reveal the fine modulation of ER Ca2+ signaling, and the activation of the store-operated Ca2+ entry (SOCE) pathway guarantees the replenishment of the ER so that the cell can be ready for response to the subsequent stimulus.To explore the above issues, CaThe animal experiments were approved by the Ethic Committee, School of Biomedical Engineering, Shanghai Jiao Tong University. All procedures strictly conformed to the humane treatment and use of animals as prescribed by the Association for Research in Vision and Ophthalmology.Carassius auratus, 15–20 cm body length). After 30 min dark-adaption, the eye was enucleated and hemisected. The retina was then isolated and cut into 8–12 pieces and incubated for 30 min at room temperature of 25°C in 5 ml Hank’s solution (see below) containing 25 U/ml papain  and 1 mg/ml L-cysteine. The retinal pieces were then kept in normal Hank’s solution at 4°C until being used (within 4 h). To obtain dissociated H1 HCs, retinal pieces were gently triturated with fire-polished glass pipettes in normal Ringer’s solution. The cell suspension was moved to a recording chamber for Ca2+ imaging recording.H1 HCs were enzymatically dissociated from retinas of adult carp : 145.0 NaCl, 5.0 KCl, 1.0 MgSO4, 2.0 CaCl2, 10.0 HEPES and 16.0 Glucose. Ca2+-free Ringer’s solution was prepared based on normal Ringer’s solution with CaCl2 omitted and 1 mM EGTA added. Caffeine was directly dissolved in Ringer’s solution according to the concentration required. Ryanodine , nifedipine, thapsigargin (TG) and 2-aminoethoxydiphenyl borate (2-APB) were prepared in dimethyl sulfoxide (DMSO) and diluted to their final concentration in Ringer’s solution (DMSO <0.5%). The pH value was adjusted to 7.4 with NaOH for Ringer’s and Hank’s solutions as well as other solutions. All drugs, unless otherwise specified, were purchased from Sigma Aldrich .Hank’s solution contained (in mM): 137.0 NaCl, 3.0 KCl, 1.0 MgSO2+]i changes were measured using a fluo-3 imaging system. Fluo-3/AM was dissolved in DMSO (1 mM stock solution) and added to the cell suspension at a final concentration of 5 µM (DMSO = 0.5%). Cell suspension was transferred into a perfusion chamber  with a cover glass bottom  and incubated at room temperature (25°C) for 15 min to allow for cell adherence and fluo-3 loading. The cells were then continuously perfused with Ringer’s solution at a flow rate of 1 ml/min for 10 min prior to recording. The volume of the perfusion chamber was about 500 µl. All drugs were applied by perfusion. At the perfusion rate employed, 1 min was needed for the drug to reach the perfusion chamber. Fluorescence measurements were performed on an upright fluorescence microscope . H1 HCs were identified by their characteristic morphology as having a round soma with 4–8 extended, subtle dendrites 2+-bound form was excited at 488 nm, and fluorescence emission at 525 nm was acquired every 2 s by a digital CCD camera . [Ca2+]i signals were presented by the fluorescence intensity F normalized to the baseline fluorescence value F0 (F/F0). For caffeine-induced [Ca2+]i responses, [Ca2+]i signals were normalized against the amplitude of the first [Ca2+]i transient. A transient peak was considered a caffeine-induced [Ca2+]i transient if it had an amplitude larger than 3×SD of data recorded in the 1 min duration prior to caffeine application.i signal initiated by ER Ca2+ release, caffeine was applied at six different concentrations  in combination with four different durations . For each of these 6×4 = 24 combinations, one group of independent experiments was conducted .Caffeine has long been used as a RyR agonist for studying RyR-mediated CaTo study the effect of duration of caffeine application, the four groups of experiments with varying durations  of caffeine application at the same caffeine concentration were compared.2+]i changes in all four groups of H1 HCs tested.In our experiments, the application of 1 mM caffeine with durations of 30, 60, 90, and 240 s induced no discernible [Ca2+]i transient(s) in a duration-dependent way  when the duration was increased from 30 to 240 s , when the duration of caffeine application was increased from 30 to 240 s , the pattern of [Ca2+]i transient could be induced no matter how long the caffeine application duration was , with the oscillatory behavior being concentration-dependent. To examine the effect of caffeine concentration on Ca2+ oscillations, experiments conducted at varying caffeine concentrations with identical duration (90 s) of caffeine application were compared  was decreased from 0.82±0.05 to 0.41±0.07  pumps 2+-increasing and removal processes determines the pattern of [Ca2+]i oscillations, which is reflected in the time interval between two adjacent transients, and the amplitude of each transient. To investigate the underlying mechanisms of caffeine-induced [Ca2+]i oscillations, we looked into the involvement of RyR activation in the initiation of the [Ca2+]i oscillations.Caffeine-induced [Ca2+]i oscillations can be initiated by Ca2+ release from the ER via RyRs. To confirm the contribution of RyRs during caffeine-induced [Ca2+]i oscillations, high concentration ryanodine (2.5 µM) was used to inhibit the Ca2+ release via RyRs 2+]i transients in an example HC  was 1.00±0.00 for the ryanodine group, which was significantly reduced as compared with 2.88±0.23  of the control group , Ca2+-free Ringer’s solution was used to abolish the putative Ca2+ influx across the plasma membrane.For each [Ca2+]i transients in the tested HC, and such response pattern was repeatable  elicited two [Ca2+]i transients, with the subsequent caffeine application inducing no measurable response  again induced [Ca2+]i response, confirming the necessity of Ca2+ influx for ER refilling following its depletion for the subsequent Ca2+ response.The results show that, in normal Ringer’s solution (control), caffeine  induced three i transients induced by caffeine  was significantly reduced as compared with control , ΔT1–2 and A2/A1 were calculated. The ΔT1–2 interval was similar to that measured in control  to 4 mM to up-regulate Ca2+ entry from the extracellular space.The above results show that caffeine-induced [Ca2+]o, three [Ca2+]i transients were evoked in an example HC  application was given in the presence of 4 mM [Caample HC . Statist2+]i response was significantly reduced when [Ca2+]o was removed, which indicates that Ca2+ influx is requested for the caffeine-induced [Ca2+]i oscillations. In carp HCs, Ca2+ entry from the extracellular space is known to be mediated by Ca2+-permeable glutamate receptors (GluRs) 2+]i are strongly correlated. Oscillations in [Ca2+]i may concomitant changes of the membrane potential. Thus, L-VGCCs, which are activated by membrane depolarization, might be involved in the caffeine-induced [Ca2+]i increase. To test the possibility of L-VGCC involvement, nifedipine, an L-VGCC antagonist, was applied 2+ entry The above results show that the caffeine-induced [Ca2+]i transients, the subsequent caffeine application also induced two [Ca2+]i transients , as compared with the effect of caffeine  alone , caffeine  elicited two [Caansients . Nifedip2+]i transient number suggested that L-VGCCs should be activated by caffeine application, and the resulting Ca2+ entry via L-VGCCs contributed to caffeine-induced [Ca2+]i oscillations. However, in the presence of nifedipine, the number of [Ca2+]i transients was significantly larger than that in Ca2+-free Ringer’s solution  2+ channels mediating SOCE are called store-operated channels (SOCs).It has been reported that in some cell types, Ca2+-free Ringer’s solution containing 5 µM thapsigargin, an irreversible SERCA inhibitor, which causes passive depletion of ER Ca2+ by inhibiting ER Ca2+ uptake via SERCA pumps 2+]o should result in an increase in [Ca2+]i, given that ER Ca2+ uptake via SERCA pumps was inhibited. As shown in 2+-free Ringer’s solution for 20 min, re-introduction of [Ca2+]o (2 mM) caused a transient increase of [Ca2+]i in the tested HC  application, 2-APB (100 µM) was applied.During the application of 3 mM caffeine, the number of Caed range , suggest2+]i oscillations were induced by prolonged application of 3 mM caffeine  was co-applied after 2 min caffeine application  was 2.22±0.15, which was significantly reduced as compared with 8.57±0.81 in normal Ringer’s solution (control)  also induced oscillatory responses in carp H1 HCs in a concentration-dependent manner – when the concentration of caffeine was increased, the time interval between the two adjacent response peaks was decreased. However, the frequency ranges of caffeine-induced [Ca2+]i oscillations observed in bullfrog sympathetic neurons were much lower when compared with that in carp H1 HCs. On the other hand, while the amplitudes of oscillatory caffeine-induced [Ca2+]i increases were basically unaltered in bullfrog sympathetic neurons during the time course of caffeine application, a decreasing tendency was observed in the amplitudes of the oscillatory caffeine-induced [Ca2+]i increases in carp H1 HCs, such differences might result from the differences in frequency ranges. At low oscillation frequencies, depleted ER might be fully refilled, so the amplitudes of the oscillatory caffeine-induced [Ca2+]i increases were basically unaltered in bullfrog sympathetic neurons, while that measured in carp H1 HCs in our experiments had a decreasing tendency.Caffeine has long been used as a RyR agonist for studying RyR-mediated Ca2+ activates Ca2+ influx across the plasma membrane (PM) 2+ influx pathway that exists in a variety of cell types, including neurons 2+ content 2+ concentration inside the lumen of the ER ([Ca2+]ER). Two types of STIM-regulated SOCs have been described: the Orai channels 2+ influx to refill depleted intracellular stores and regulate cellular processes 2+ entry in HCs.SOCE is a process by which the depletion of ER Ca2+ entry via SOCs was mainly transported into the ER 2+]i oscillations, ER refilling occurred during the interval between two adjacent [Ca2+]i transients, during which [Ca2+]i was relatively low, suggesting that the majority of Ca2+ entering the cytoplasm via SOCs was transported into the ER. This phenomenon reflected a coupling between the SOCs and SERCA pumps in carp H1 HCs.SERCA is one of the proteins identified as a part of the SOC macromolecular complex 3Rs In our experiments, 2-APB was used to inhibit SOCE –5. Altho2+]o2+]o in a concentration dependent manner 2+]o was remained constant at a level of 2 mM, and a small fraction of HGJ channels might be active. However, the application of 2-APB (100 µM) had no significant effect on the basal level of [Ca2+]i (data not shown), suggesting that even if HGJ channels were active, the inward HGJ channel currents had little contribution to [Ca2+]i responses observed in the present study. Outward HGJ channel currents with amplitudes larger than the inward currents can be evoked by membrane depolarization (beyond 10 mV) 2+ oscillations were abolished by inhibiting either Ca2+ influx or ER Ca2+ release. Therefore, it is unlikely that 2-APB abolished Ca2+ oscillations by inhibiting outward HGJ channel currents.HGJ channels are expressed in fish retinal HCs, and are involved in negative feedback from HCs to cones 3Rs, no study suggests the expression of IP3Rs in carp retinal HCs. Besides, in experiments shown in 3Rs which mediate Ca2+ release from the ER were not involved in the [Ca2+]i increase observed after re-addition of extracellular Ca2+. Therefore, it is unlikely that 2-APB abolished [Ca2+]i signals observed in our experiments by inhibiting IP3Rs.In regard to IPTaken together, despite the fact that 2-APB affects channels other than SOCs, we can still make an inference that there exists a SOCE pathway in carp H1 HCs, which reloads the ER after its depletion.2+]i changes in H1 HCs, and a single [Ca2+]i transient was evoke by high concentration (40 mM) caffeine, while [Ca2+]i oscillations were induced by caffeine at intermediate concentrations (3 to 20 mM). The present results indicated that each [Ca2+]i transient was initiated by RyR activation, and ER refilling, which was required for the generation of subsequent [Ca2+]i transients, was depending on SOCE. Hence RyRs and SOCs are likely the two major components for caffeine-induced [Ca2+]i oscillations.Our results showed that caffeine at low concentration (1 mM) elicited no i response pattern on caffeine concentration observed in our present study. When caffeine was applied at low level (1 mM), the RyR Po was very small o significantly increased and approaching 1.0 2+ after the initial release, therefore only one Ca2+ transient was generated.Researches on caffeine effect on single RyR channel found that RyR open probability , short interval between two adjacent [Ca2+]i transients resulted in inadequate ER replenishment, thus the amplitudes of the oscillatory Ca2+ transients were gradually decreased until the ER was finally running out of Ca2+.The amplitudes of the subsequent [Ca2+ is an important intracellular messenger in carp HCs. [Ca2+]i increase is involved in many HCs cellular functions, including modulation of GABA transporter currents 2+ signaling requires the expression of many Ca2+-related components to create a wide range of spatially and temporally distributed signals Similar to other cell types, Ca2+), leading to HC depolarization, VGCC activation, CICR from the ER and an increase in [Ca2+]i2+]i is cytotoxic 2+]i must be brought back to the basal level so that the cell can be ready for response to the subsequent stimulus. After the initial [Ca2+]i increase, AMPA receptor desensitization 2+ influx, regulating [Ca2+]i increases and CICR from the ER. Hence, oscillatory [Ca2+]i responses, such as that observed in the present study, might be one of the strategies that HCs adopt in exposure to tonic glutamate input, so as to prevent cytotoxicity, as well as regulating [Ca2+]i by the rate and duration of photoreceptor glutamate release.In the vertebrate retina, glutamate is continuously released from photoreceptors in the dark, thus GluRs on HCs can be tonically activated. Activated ionotropic GluRs mediated cation influx (including Ca2+]i are strongly correlated, we infer that [Ca2+]i oscillations can be induced under physiological conditions. But the frequency ranges of the membrane potential oscillations were between 1.5 and 3.5 Hz, much higher than the [Ca2+]i oscillations recorded in our experiments. In our experiments, bath application of caffeine affects RyRs in isolated H1 HCs, leading to the generation of global intracellular Ca2+ signals. While in the intact retina, HCs receive input from synapses of rods and cones, producing localized Ca2+ signals. Depletion of the ER in a restricted area takes shorter time to refill, while depletion of the whole ER as observe in the current study takes longer to refill, this might account for the differences in oscillation frequencies observed in HCs in the intact retina and isolated HCs.Oscillations of the membrane potential in response to a brief light flash have been recorded from HCs in the intact retina 2+]i oscillations in carp H1 HCs is the repetition of the following two steps: ER Ca2+ release and subsequent ER refilling. Ca2+ release from the ER is mediated by RyRs, while ER refilling depends primarily on Ca2+ influx via SOCs. Hence the SOC is an essential component for ER refilling, and its activation is required for the maintenance of [Ca2+]i oscillations. The ER is the primary intracellular reservoir of Ca2+ and a major source of [Ca2+]i elevation, and it is involved in many cellular processes. The existence of SOCs in H1 HCs and the coupling between SOCE and ER refilling guarantee the efficient replenishment of the ER so that the cell can be ready for response to the subsequent stimulus. Thus SOCs are essential for the fulfilling of normal physiological functions of carp H1 HCs.Generally, caffeine-induced [Ca


Grana and starch are major functional structures for photosynthesis and energy storage of plant, respectively. Both exhibit highly ordered molecular structures and appear as micrometer-sized granules inside chloroplasts. In order to distinguish grana and starch, we used multiphoton microscopy, with simultaneous acquisition of two-photon fluorescence (2PF) and second harmonic generation (SHG) signals. SHG is sensitive to crystallized structures while 2PF selectively reveals the distribution of chlorophyll.Three distinct microstructures with different contrasts were observed, i.e. “SHG dominates”, “2PF dominates”, and “SHG collocated with 2PF”. It is known that starch and grana both emit SHG due to their highly crystallized structures, and no autofluorescence is emitted from starch, so the “SHG dominates” contrast should correspond to starch. The contrast of “SHG collocated with 2PF” is assigned to be grana, which exhibit crystallized structure with autofluorescent chlorophyll. The “2PF dominates” contrast should correspond to stroma thylakoid, which is a non-packed membrane structure with chrolophyll. The contrast assignment is further supported by fluorescence lifetime measurement.We have demonstrated a straightforward and noninvasive method to identify the distribution of grana and starch within an intact leaf. By merging the 2PF and SHG images, grana, starch and stroma thylakoid can be visually distinguished. This approach can be extended to the observation of 3D grana distribution and their dynamics in living plants. Inside a chloroplast, thylakoid has two structural forms. One is stroma thylakoid (stroma lamellae), which is a membrane-bound structure embedded into the stroma of chloroplast. The other is granum, which is a well-packed stack of thylakoids  and scanning electron microscopy (SEM) -6. As exOptical microscopy, on the other hand, exhibits much lower spatial resolution , but it can be used to view cells that are living and functioning. Conventional compound microscopes are not capable of producing a clear three-dimensional view in thick biological tissues. By combining a confocal pinhole with laser scanning microscopy, confocal microscopy provides excellent optical sectioning capability. In the late 1980s, confocal microscopy was adopted to study the inner structures of chloroplasts in living and intact plants based on the autofluorescence from chlorophyll ,8. HowevNonlinear optical microscopy has become an important tool for imaging biological samples -14. TherIn the case of SHG, laser light is focused on a sample to generate frequency-doubled light. During SHG process, two incident photons are annihilated and a new photon is generated. Due to energy conservation, the energy, and thus frequency, of the new photon is twice of that of the annihilated photons, as shown in Figure The aim of this paper is to demonstrate that nonlinear optical microscopy is a noninvasive method to identify the distribution of grana, stroma thylakoid, and starch granules within a live intact leaf. No complicated sample preparation or darkroom process is required. Through simultaneous acquisition of 2PF and SHG signals, these cellular organelles can be visually identified. This novel approach can be used in the field of botanical evolutionism, and provides a dynamic imaging observation in the growth of the plants.Figure In Figure In addition, an axial image series  structure, so SHG image reveals the distribution of grana and starch granules, which are the only two organelles inside a chloroplast that emit SHG. By combining both 2PF and SHG modalities, the signal from grana, starch, and stroma thylakoid, can be well separated visually, as summarized in Table One possible ambiguity lies in the interpretation of overlapped SHG and 2PF. Our current explanation is that the overlapping reflects the coexistence of chlorophyll autofluorescence and stacked structures, i.e. grana. However, it is possible that the structure we assigned to be a granum might be a starch granule surrounded by stroma thylakoid. Here we describe the reason why the latter interpretation is more unlikely.Let's examine Figure In order to confirm the discrimination among stroma thylakoid, grana, and starch granules, we have performed additional fluorescence lifetime imaging measurement. It is known that fluorescence of chloroplast comes mainly from photosystems (PS), including PSI and PSII. About 85% of PSII is located in grana, while PSI is dominating in stroma thylakoid ,30. NeveWe have demonstrated a noninvasive method to identify the distribution of grana and starch inside a live mesophyll cell without any special specimen labeling or handling. The method is based on the combination of SHG and 2PF contrast in a multiphoton microscope. There are two types of SHG structures inside a chloroplast of a plant cell. One is collocated with strong 2PF, and the other is complementary to 2PF. The former correspond to grana while the latter correspond to starch. For those regions with only 2PF but no SHG, they represent the distribution of stroma thylakoid. By merging 2PF and SHG images, the grana, starch granules, and stroma thylakoid can be visually distinguished by different colors of yellow (green + red), green, and red, respectively. The structure identification is further proved by fluorescence lifetime measurements. The nonlinear nature of the multiphoton process provides useful intrinsic optical sectioning capability and is less likely to cause damage in live sample, enabling observation of organelle dynamics during plant growth. Our technique will be useful to study granal structural variation among different plant specie , and canMacrothelypteris torresiana (Gaud.) Ching, which belongs to shaded plants with large grana [The leaf we used here was detached from a fresh ferns, ge grana -37. The -6 sec, and the acquisition time for one image (256 × 256 pixels) is about 0.6 sec. The excitation light was focused onto the specimen through the microscope objective lens . The average laser power at sample position is about 60 – 70 mW. The 2PF signals were epi-collected by the same objective while the SHG signals were collected by a condenser in the forward direction. Two identical photomultiplier tubes  with coolers were respectively placed in the forward and backward path to detect SHG and 2PF signals. There is a dichroic mirror  inside FV300 to reflect IR and to allow the transmission of the 2PF signals. Additional color filters  were placed in front of each PMT to ensure that laser is appropriately blocked. Filters for SHG  and for 2PF  are inserted before corresponding PMTs to ensure only SHG and 2PF signals were recorded.The experimental setup is shown in Figure For fluorescence lifetime measurement, the excitation and scanning systems are the same as above, but the detection part becomes a photon-counting PMT  in the forward direction, equipped with a time-correlated single photon counting system . A high-speed photodetector synchronize the laser repetition rate to the photon counting system. During lifetime measurement, corresponding filters are placed in front of the photon counting PMT to allow either 2PF or SHG detection without cross talk.MYC, GYZ, KCC, PCW, and TYH performed the experiments. MYC analyzed the data and wrote the manuscript with SWC. TML and SWC supervised the project and instruments. All authors read and approved the final manuscript.Z-stack merge image of Macrothelypteris torresiana (Gaud.) Ching leaf. The chloroplasts are filled with 2PF (red) overlapped with discrete SHG (green) spots.Click here for file


It is also recognized that sympathetic nerve-mediated contractions of the vas deferens can be modulated by acetylcholine (Ach), however there is considerable disagreement in the literature regarding the precise contribution of cholinergic nerves to contraction of the vas deferens. In this study we examined the effect of cholinergic modulators on electric field stimulation (EFS)-evoked contractions of rabbit vas deferens and on cytosolic Ca2+ levels in isolated vas deferens smooth muscle cells (VDSMC). The sustained component of EFS-evoked contractions was inhibited by atropine and by the selective M3R antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP). EFS-evoked contractions were potentiated by Ach, carbachol (Cch), and neostigmine. The sustained phase of the EFS-evoked contraction was inhibited by prazosin, an α1-adrenoceptor antagonist and guanethidine, an inhibitor of noradrenaline release, even in the continued presence of Ach, Cch or neostigmine. The soluble guanylate cyclase (sGC) inhibitor, 1H-Oxadiazoloquinoxalin-1-one enhanced the amplitude of EFS-evoked contractions and reduced the inhibitory effects of 4-DAMP. Isolated VDSMC displayed spontaneous Ca2+ oscillations, but did not respond to Cch. However, the α1-adrenoceptor agonist, phenylephrine, evoked a Ca2+ transient and contracted the cells. These data suggest that EFS-evoked contractions of the rabbit vas deferens are potentiated by activation of M3 receptors and reduced by activation of a sGC-dependent inhibitory pathway.Stimulation of intramural nerves in the vas deferens of many species yields a classical biphasic contraction comprised of an initial fast component, mediated by P The vasa deferentia were removed and placed in Krebs’ solution for further use.All experiments were approved by the Dundalk Institute of Technology (DkIT) Animal Care and Use committee and were in accordance with EU Directive 2010/63/EU. New Zealand White rabbits (16–20 weeks old) were humanely killed with a lethal injection of pentobarbitone . EFS was applied via two platinum electrode wires  by a MultiStim system-D330 stimulator , which delivered 0.3-ms pulses of 20 V  at a frequency of 4 Hz for 1 min.Longitudinal segments of vas deferens (˜8 mm in length) were dissected from the mid section of the tissue. These were mounted in water-jacketed organ baths maintained at 37°C and bathed with Krebs’ solution bubbled with 95% O3 pieces and stored in Ca2+-free Hanks solution for 30 min prior to cell dispersal. Tissue pieces were incubated in dispersal medium containing (per 5 mL) of Ca2+-free Hanks solution (see solutions): 15 mg collagenase (Sigma type 1A), 1 mg proteinase (Sigma type XXIV), 10 mg bovine serum albumin , and 10 mg trypsin inhibitor (Sigma) for 10–15 min at 37°C. Tissue was then transferred to Ca2+-free Hanks solution and stirred for a further 15–30 min to release single smooth muscle cells. These were plated in Petri dishes containing 100 μmol/L Ca2+ Hank's solution and allowed to settle in glass bottomed Petri dishes until they had stuck down. They were then incubated in 0.4 μmol/L fluo-4/AM  for 6–8 min in the dark at room temperature before being studied. During experiments, the dish containing the cells was continuously perfused with Hanks solution at 36 ± 1°C. Additionally, the cell under study was continuously superfused by means of a custom built close delivery system with a pipette of tip diameter 200 μm placed ˜300 μm from the cell. The Hanks solution in the close delivery system could be switched to a drug-containing solution with a dead-space time of less than 5 sec.Vas deferens segments were cut into 1 mmμm) coupled to a Nipkow spinning disk confocal head . A krypton-argon laser  at 488 nm was used to excite the fluo-4, and the emitted light was detected at wavelengths >510 nm. Experiments were performed using a 60× objective  resulting in images of pixel size 0.266 × 0.266 μm. Images were acquired at 15 frames per second. Background fluorescence from the camera, obtained using a null frame, was subtracted from each frame to obtain “F.” F0 was determined as the minimum fluorescence measured between oscillations under control conditions. To obtain post hoc pseudo line-scan images for display in figures, a one pixel thick line was drawn centrally through the entire length of the cell and the “reslice” command in Image J  was invoked. Plots of F/F0 were obtained from the post hoc line-scan by drawing a rectangle around the entire area of the line-scan image and plotting the intensity profile in Image J. ΔF/F0 refers to the measurement of the change in Ca2+ levels from basal to peak.Cells were imaged using an iXon 887 EMCCD camera , KH2PO4 (0.4), NaH2PO4 (0.3), MgCl2·6H2O (0.5), CaCl2·2H2O (1.8), MgSO4 (0.4), Hepes (10.0). pH to 7.4 using NaOH. pH adjusted to 7.4 with NaOH; Ca2+-free Hanks solution : NaCl (125), KCl (5.36), glucose (10), sucrose (2.9), NaHCO3 (15.5), KH2PO4 (0.44), Na2HPO4 (0.33), N-[2-Hydroxyethylpiperazine]-N′-[2-ethanesulfonic acid]  pH adjusted to 7.4 with NaOH. Krebs’ solution: NaCl (120), KCl (5.9), NaHCO3 (1.2), glucose (5.5) CaCl2 (2.5), MgCl2 (1.2) pH maintained at 7.4 by bubbling with 95% O2–5% CO2.The solutions used were of the following composition (mmol/L): Normal Hanks Solution: NaCl (125.0), KCl (5.4), Glucose (10.0), Sucrose (2.9), NaHCOα,β-methylene ATP, tetrodotoxin (TTX), and phenylephrine (PE). All drugs were purchased from Sigma, except 4-DAMP and ODQ which were supplied by Tocris and TTX which was supplied from Abcam Biochemicals . Drugs were prepared as concentrated stock solutions in the appropriate solvent before being added to the tissue bath or reservoir to achieve the stated final concentration.Atropine, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), 1H-Oxadiazoloquinoxalin-1-one (ODQ), Prazosin, Guanethidine, Neostigmine, Ach, Cch, t-test (two tailed test) taking the P < 0.05 level as significant. Multiple comparisons were analyzed using one-way analysis of variance (ANOVA), with a Bonferroni post hoc test. Throughout, “n” refers to the number of tissue segments or cells in each experimental series. These were obtained from a minimum of three animals.Summary data are presented as the mean ± SEM and statistical differences in experiments were compared using Student's paired μmol/L), from 16.8 ± 1.8 and 10.5 ± 2.2 mN under control conditions to 0.28 ± 0.28 and 0.19 ± 0.17 mN, respectively . To test if the EFS-evoked contractions involved activation of muscarinic acetylcholine receptors, the effect of the nonselective MR antagonist, atropine (1 μmol/L) was examined. P < 0.05) and the transient contraction was not affected by the drug . These summary data are plotted in 3R antagonist 4-DAMP (30 nmol/L). Thus, the sustained contraction was greatly reduced, from 17.3 ± 2.9 to 2.8 ± 0.7 mN in 4-DAMP , but the transient contraction was not significantly reduced .EFS of isolated rabbit vas deferens segments yielded biphasic contractile responses that were reproducible when repeated at 20-min intervals. Both the transient and the sustained phase of the contraction were inhibited by TTX (1 μmol/L), the MR agonist Cch (1 μmol/L) and the acetylcholinesterase inhibitor neostigmine (1 μmol/L) on EFS-evoked contractions of vas deferens. Application of these agents did not evoke sustained tonic contractions. However, as can be seen from the respective representative records in n = 19, P < 0.05), however, it was notable that neostigmine produced a smaller enhancement of the transient contraction compared to Ach or Cch. In order to test if these potentiation effects were due to direct stimulation of postjunctional Ach receptors, or indirectly, via release of NA acting on α1-adrenoceptors, we repeated the experiments, described in α1-adrenoceptor antagonist, prazosin (1 μmol/L). Under control conditions application of prazosin abolished the sustained phase of contraction from a mean of 8.6 ± 2.5 mN  as previously demonstrated by Sneddon et al. . Prazosin abolished the sustained phase of EFS contractions and also reduced the amplitude of the transient phase that was enhanced by Cch . P < 0.05, n = 6). The residual transient contractions that remained in the presence of prazosin were abolished by application of the desensitizing P2X receptor agonist α,β-methylene ATP  suggesting that the transient component of the EFS contraction was mediated by P2X receptors. Taken together, these data suggest that potentiation of the second component of contraction by Ach, involves activation of α1-adrenoceptors, possibly by stimulating release of NA from sympathetic nerves. This idea was tested further by investigating if the stimulatory effects of Ach were affected by inhibition of NA release from sympathetic nerves using guanethidine.Next, we examined the effect of exogenous bath application of Ach  on EFS-induced contractions that were potentiated by Ach. Guanethidine significantly inhibited both components of contraction in the presence of Ach . However, the effect on the sustained element was notably greater than the transient contractions, which were not significantly different from those observed before addition of Ach (P > 0.05). Similar inhibitory effects of guanethidine were observed in response to stimulation by Cch (n = 9) and neostigmine (n = 9). These results further suggest that the excitatory effects of Ach were brought about by release of NA from sympathetic nerves.n by Cch , n = 9 aα1-adrenoceptor agonist, PE on Ca2+ levels in freshly isolated rabbit VDSMC. μmol/L) and Cch (1 μmol/L) for 10 sec to the same cell and 2+ oscillations, along with more intense global oscillations that occurred at a lower frequency and were associated with slight contractions of the cell. When PE was applied it caused an intense Ca2+ transient that induced a robust contraction of the cell. Following wash-out, the cell began to relax and the spontaneous activity returned. Next, Cch was applied, however, there was no discernable response to the drug. Finally, following a ˜50 sec interval, PE was reapplied to the cell, evoking a sharp rise in Ca2+, similar to the initial response. 2+ transient with a mean amplitude of 6.3 ± 1.1 ΔF/F0. Cch was without effect in every cell.The data above suggested that the stimulatory effects of Ach on EFS-evoked contractions were unlikely to be mediated by direct activation of postjunctional Ach receptors on vas deferens smooth muscle cells (VDSMC). To examine this issue more closely we examined the effect of Cch and the l-NAME, indicating an inhibitory role for NO in the rat vas deferens. NO is known to relax smooth muscle by activating the enzyme soluble guanylate cyclase (sGC). Therefore, we examined if a sGC-dependent mechanism affected EFS-evoked contractions in this study by examining the effect of ODQ, a sGC inhibitor, on EFS-induced contractions. μmol/L). ODQ dramatically enhanced the sustained element of the contraction and this effect was repeatable in 16 preparations in which the mean amplitude of the sustained contraction increased from 10.4 ± 1.6 mN under control conditions to 21.7 ± 2.7 mN in ODQ, n = 16, P < 0.05). ODQ also produced a small, but significant enhancement of the transient contraction amplitude from 8.7 ± 1.4 mN before, to 11.2 ± 1.6 mN during the addition ODQ . These data suggest that the amplitude of EFS-evoked contractions in the vas deferens may be negatively regulated by activation of sGC.The data presented thus far suggest that Ach potentiates EFS-induced contractions of rabbit vas deferens by stimulating release of NA from sympathetic nerves. However, a puzzling aspect of the study was; why, in the presence of atropine and 4-DAMP, was there not a contractile response resulting from direct stimulation of sympathetic nerves? We speculated that one potential reason for this could be that EFS also activates an inhibitory pathway that opposes contraction, such that in the absence of the excitatory Ach effects, this inhibitory effect dominates. Studies by Gur et al.  and da Sμmol/L). P < 0.05, n = 6). Following wash-out of 4-DAMP, the cell was exposed to ODQ, which enhanced the contractile response  on EFS-induced contractions on the same preparation, before, and during the presence of ODQ  Contractions were potentiated by Ach, Cch, and neostigmine, and these responses were inhibited by prazosin, an α1-adrenoceptor antagonist and guanethidine, an inhibitor of NA release; (3) The sGC inhibitor, ODQ enhanced the amplitude of EFS-evoked contractions and reduced the inhibitory effects of 4-DAMP; (4) Isolated VDSMC displayed spontaneous Ca2+ oscillations, but did not respond to Cch. However, the α1-adrenoceptor agonist, PE evoked a Ca2+ transient and contracted the cells.This study examined the effect of Ach on EFS-evoked contractions of rabbit vas deferens. EFS of the rabbit vas deferens produces robust contractions that have the classical biphasic contraction, consisting of an initial transient and secondary sustained component  (The dramatic inhibitory effects of atropine and 4-DAMP on the sustained phase of contraction were surprising because it has been known for many years that the tonic phase of the biphasic contraction is brought about by activation of  Ventura . The sim Ventura  it seemss (M3Rs) . Yet, ifl-NAME, indicating an inhibitory role for NO in the EFS response  by stimulating production of guanosine 3′,5′-cyclic monophosphate (Friebe et al. 2+ transient or a contraction we speculate that a prejunctional mechanism, involving activation of M3Rs, is most likely to account for the results of this study (The amplitude of EFS-evoked contractions of rabbit vas deferens in this study was potentiated by Ach, Cch, and neostigmine. Similar findings were reported for other species, including rat (Chung and Freer is study . Howeveris study  reportedα2-adrenoceptors. However, both prazosin and guanethidine have also been shown to reduce release of ATP in guinea-pig vas deferens (von Kügelgen and Starke A puzzling aspect of this study was that, although the transient element of the EFS-evoked contractions was unaffected by atropine and 4-DAMP, they were potentiated by exogenous application of Ach, Cch, and neostigmine. However, these effects were less consistent than those on the sustained component, as neostigmine did not always induce an enhancement of the transient phase. Furthermore, these effects were also partly reversed by prazosin and guanethidine suggesting that they could be due to release of NA. Similar effects were reported by Miranda and Wolstenholme ; who con3Rs that enhance contraction and an inhibitory sGC-dependent pathway that inhibits contraction.This study demonstrates that biphasic neurogenic contractions of rabbit vas deferens smooth muscle are more complex than first thought. The amplitude of EFS-evoked contractions is not just dependent upon direct release of ATP and NA from sympathetic nerves, but results from a balance between activation of MNone declared.