Currently the mechanisms of Ca2+ signals in astrocytes are not fully clear. Here, we present a computational model to specify the relative contributions of different Ca2+ flows between the extracellular space, the cytoplasm and the endoplasmic reticulum of astrocytes to the generation of spontaneous Ca2+ oscillations (CASs) and cortical spreading depression (CSD)-triggered Ca2+ waves (CSDCWs) in a one-dimensional astrocyte network. This model shows that CASs depend primarily on Ca2+ released from internal stores of astrocytes, and CSDCWs depend mainly on voltage-gated Ca2+ influx. It predicts that voltage-gated Ca2+ influx is able to generate Ca2+ waves during the process of CSD even after depleting internal Ca2+ stores. Furthermore, the model investigates the interactions between CASs and CSDCWs and shows that the pass of CSDCWs suppresses CASs, whereas CASs do not prevent the generation of CSDCWs. This work quantitatively analyzes the generation of astrocytic Ca2+ signals and indicates different mechanisms underlying CSDCWs and non-CSDCWs. Research on the different types of Ca2+ signals might help to understand the ways by which astrocytes participate in information processing in brain functions.Astrocytes participate in brain functions through Ca In addition to the Ca2+ released from internal stores, Ca2+ influx from the extracellular fluid is also reported to be needed to generate Ca2+ oscillations 2+ influx 2+ is not required for the occurrence of Ca2+ oscillations 2+ released from the ER is usually considered to be the key factor in the generation of Ca2+ waves which are induced by ATP or IP32+ waves are triggered by cortical spreading depression (CSD) 2+ signals suggest that the underlying mechanisms in the generation of Ca2+ oscillations and Ca2+ waves are still unclear.Many experiments have suggested that Ca2+ oscillations (CASs) and CSD-triggered Ca2+ waves (CSDCWs) have been reported in the same experiments 2+ signals. However, these models either focused just on CASs 2+ waves induced by ATP or IP3 but not by CSD (non-CSDCWs) 2+ flows within the astrocytes, for example, the Ca2+ released from CICR and the Ca2+ uptaken into the ER, were neglected in their model 2+ from the ER for the generation of different types of Ca2+ waves (CSDCWs and non-CSDCWs) could not be fully explained by these models.Interestingly, spontaneous Ca2+ flows between the extracellular space, the cytoplasm and the ER of astrocytes to the generation of these Ca2+ signals. We first explored the mechanisms for the generation of CASs and CSDCWs, and then investigated the interactions between CASs and CSDCWs, and finally addressed the transition from CASs to CSDCWs. Our results quantitatively analyze the generation of astrocytic Ca2+ signals and indicate different mechanisms underlying CSDCWs and non-CSDCWs.In the present study, we investigated CASs and CSDCWs in a one-dimensional astrocyte network by an expanded version of our previous model, which simulated the VGCCs-mediated CASs µm. In a single-cell context, three compartments were considered, including the extracellular space (ECS), the intracellular space (ICS), and the ER internal space, as seen in 3 from cytosol to cytosol through gap junctions  was converted into flux to calculate its contribution to the increase of Ca2+ in astrocytes:Vastro is the volume of an astrocyte.2+ in astrocytes is partly discharged into the ECS by the Ca2+ pump, and the calculation is adapted from gpump is the membrane conductance of the pump and additionally, the current in Eq. (6) was also converted into flux:CaLext is the rate of Ca2+ efflux from astrocytes.2+ flows through the membrane of ER included Ca2+ released from CICR, Ca2+ uptaken into the ER via the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) and leak flux through the membrane of ER, which are described as JCICR, JSERCA and Jleakics in Eqs. (S1)-(S3) of 2+ in the astrocytic cytosol were expressed asDCai is the diffusion coefficient for Ca2+ in the cytosol and Δ is the Laplace operator. The first term on the right of Eq. (9) represents the diffusion of Ca2+ in the ICS. Accordingly, Ca2+ in the ER (CaER) was determined byAs shown in Cao were neglected owing to its slight changes during Ca2+ oscillations in previous models. However, Cao changes dramatically during CSD 2+ in the ECS was modeled byDCao is the diffusion coefficient for Ca2+ in the ECS. Ko represents the K+ concentration in the ECS. Mca is a constant, Komax is the maximal Ko during CSD, and Caorest is Cao at resting state. The first term on the right of Eq. (11) represents the diffusion of Ca2+ in the ECS. The last term on the right of Eq. (11) describes the decrease of Cao during CSD, and its precise mechanism needs to be further explored.3 is a known intracellular messenger, which can bind to the IP3R to cause Ca2+ to flow out of the ER. In a single-cell context, IP3 in the ICS (IP3) was catalyzed by phospholipase C (PLC) as defined in Eq. (S4) of 2+ waves in astrocytes are mediated following the transfer of IP33 through gap junctions. Following IP3 in astrocyte i due to the gap junction with astrocyte j isi, j are indices of adjacent astrocytes, and γ is the coupling strength. Combining the diffusion of IP3 inside the cells 3 in astrocyte i was calculated asIP3D is the diffusion coefficient for IP3 in the ICS. proIP represents the production of IP3 and IPdeg represents its degradation, as seen in Eq. (S4) of IP2+ waves. Because high extracellular K+ is required for the propagation of CSD Ko was described as the classical diffusion-reaction equation:DKo is the diffusion coefficient for K+ in the ECS, and the first term on the right of Eq. (14a) represents the diffusion of K+ in the ECS. f(Ko) describes the reaction process of Ko, which is adapted from MKK is a rate constant, Korest is Ko at the resting level, and Kθ is the threshold for the triggering of CSD. The first term on the right of Eq. (14b) meets the following requirements: maintaining homeostasis at the resting level, triggering explosive subsequent growth in Ko when Ko is higher than the threshold Kθ, and preventing Ko rising when Ko is beyond the ceiling Komax Ko. Rk, which restores Ko to the normal level, was modeled by:MKR and MR are constants.CSD causes neurons and astrocytes to depolarize and greatly changes the ion concentration KoKo would partly be untaken by astrocytes. The K+ concentration in the astrocytes (Ki) was calculated as.MKi and Mio are rate constants. Kirest is Ki at the resting level, Kiθ is the threshold for the fast elevation of Ki, and Kimax is the ceiling Ki during CSD. The first term on the right of Eq. (16) follows the formalism of Ko to maintain cytosol K+ homeostasis, the senond term is used to detect the change in Ko and is assumed to be effective only when Ko is beyond the threshold for the triggering of CSD, and the third term represents the discharge of Ki.AKi and Arr are constants. The first term on the right of Eq. (17) represents the discharge of Ki, and the second is a decay term.Astrocytes are reported to be fast buffers and play an important role in the clearance of excess + Nernst potential is close to Vm; therefore, in this study, the K+ Nernst potential was used to approximate Vm by adding a modulation factor, which was chosen based on previously experiments zK is the valence of K+ and ε is the modulation factor.The astrocytic membrane potential is a complicated parameter to calculate in computational models. To avoid these complex calculations, a simplified method was adopted. It is well known that the KN astrocytes, where N ranged from 1 to 100. All of the computations and visualizations of this model were implemented in the Matlab environment . The Crank-Nicholson algorithm was used to solve the differential equations The chain model consisted of 32+ oscillated with the amplitude of 0.63×10−3 mM and the frequency of 0.0044 Hz, which were consistent with the experimental results 2+ oscillations, measured at the half-amplitude level, was 21 s. In the ER  of 2+ oscillations, and oscillations cannot occur without Ca2+ released from the ER 2+ into the ER (reducing MSERCA in Eq. (S2) of 2+ dynamics evolve into small oscillations before finally disappearing, as seen in gVGCC, was reduced to simulate the VGCCs inhibition. After the reduction, the frequency of CASs decreased  was elevated to 12 mM to evoke a CSD. CSD would cause the local extracellular K+ to increase further and spread to the surrounding KoKo was significantly increased and spread in this model after locally increasing extracellular K+ concentration, suggesting the emergence of CSD, and then Ki was also increased to partly clear the excess K+ in the ECS  was increased gradually to explore the transition from CASs to CSDCWs in single astrocytes. It shows that increasing Ko would induce the elevation of Cai and the generation of CASs in the astrocytes near to the stimulation site (Ko caused an earlier onset time for oscillations compared to the control (the onset of CAS under the control condition is marked with an blue arrow). By increasing Ko in the present of CASs facilitated the further elevation of Cai and the elevated Cai deferred the occurrence of successive oscillations  2+ waves in astrocytes are usually thought to be induced by ATP or IP3, and they depend on Ca2+ released from internal stores 2+ waves was represented as non-CSDCWs in this study. There are at least three differences between the CSDCWs and non-CSDCWs. First, after depletion of internal stores, non-CSDCWs cannot be generated 2+ efflux from the internal stores, CSD can cause astrocytes to depolarize, which opens the VGCCs, and Ca2+ influx via VGCCs is able to generate Ca2+ waves. It suggests that the generation of CSDCWs depends primarily on Ca2+ influx via VGCCs. VGCCs have been reported to be not physiologically relevant for intracellular Ca2+ signals 2+ influx coupled to depolarization was recorded during CSDCWs 2+ waves still spread but with a significantly reduced amplitude and speed 2+ flows uncoupled to depolarization. In this study, we only simulated CSDCWs, focusing on the voltage-gated Ca2+ influx pathway, and this might explain why the amplitude of CSDCWs was not strongly attenuated after depletion of internal stores compared to the experiment data Ca2+ in the cytosol to a similar level. As a result, it took astrocytes equivalent time to recover from the effect of CSD, which might determine the latency to the onset of following CASs. Locally increasing Ko would cause astrocytes to depolarize and increase Ca2+ influx via VGCCs, which facilitated the occurrence of CASs and the elevation of Cai. Meanwhile, the onset of subsequent Ca2+ oscillation was postponed as the elevation of Cai. This is because that in addition to the increased amplitude of Cai caused by increasing Ko, the duration of Cai was also increased. As a result, astrocytes needed more time to recover from the elevation of Cai, and the subsequent oscillations came later.We investigated the interactions between CASs and CSDCWs, and showed that CSDCWs depressed CASs and CASs reappeared after the pass of CSDCWs, which was consistent with previous experiments 2+ signals in astrocytes are quite variable in the spatiotemporal organization. CASs in cortical layer 2/3 show infrequent synchronous pattern, whereas in layer 1 CASs are frequent asynchronous 2+ entry from the extracellular space as well as Ca2+ release from internal stores. VGCCs, which are expected to play significant functional roles in Ca2+ influx in astrocytes 2+ transients. According to our study, voltage-gated Ca2+ influx had a great contribution to CSDCWs, and might play a role in triggering CASs. However, the distribution of VGCCs expression in the brain tissue is not uniform. Experiments show apparent lack of VGCCs in rat hippocampus and visual cortex 2+ signals among astrocytes.Ca2+ entry via the external medium has also been found to contribute to CASs 2+ flows to the generation of astrocytic Ca2+ signals, and showed that voltage-gated Ca2+ influx played a role in regulating the frequency of CASs and might be important for initiating CASs 2+ stores is reported to block non-CSDCWs 2+ stores have been depleted CSDCWs could still propagate. Furthermore, we showed that the propagation of CSDCWs after depleting internal Ca2+ stores was due to voltage-gated Ca2+ influx, indicating different mechanisms underlying CSDCWs and non-CSDCWs. Future research should focus on the different mechanisms underlying astrocytic Ca2+ signals.Experiments show that CASs are based on the process of CICR 2+ dynamics implements the frequency modulation-encoding oscillations 2+ influx caused by the depolarizing stimulus with high extracellular K+. Frequency modulation-encoding oscillations are mediated by the process of CICR 2+ influx rather than the process of CICR, frequency modulation-encoding oscillations would not determine the transition from CASs to CSDCWs. It will be interesting to study the interactions between non-CSDCWs and CSDCWs, because that non-CSDCWs compromise a large number of synchronized astrocytes, which might affect the incidence of CSDCWs. However, we failed to elicit non-CSDCWs, which might be due to that in our model the internal Ca2+ dynamics does not implement the frequency modulation-encoding oscillations and that ATP diffusion in the ECS is not considered. Ca2+ in non-CSDCWs originates mainly from internal stores of astrocytes 2+ in CSDCWs, according to our study, originates mainly from Ca2+ influx, and Ca2+ in internal stores also contributes to CSDCWs. Base on these findings, non-CSDCWs seem not to suppress the incidence of CSDCWs but to change their properties. This might be supported by the experiments that after depletion of the internal Ca2+ stores, CSDCWs could still be recorded but the amplitude of Ca2+ signal was reduced A recent study has simulated the transition from single CASs to non-CSDCWs, and the results suggested that long-distance non-CSDCWs are favored when the internal Ca+ Nernst potential to approximate the membrane potential, but add a modulation factor. The value of the modulation factor was chosen based on the considerations: after adding the modulation factor, astrocyte membrane potential is within the range where CASs would occur 2+ recording from astrocytes in vivo shows that astrocytes were either quiescent or responded with a few Ca2+ transients 2+ transients occurred more frequently in slices prepared at 28°C 2+ events between the in vivo and in situ results 3R channels or the increased activity of SERCA at higher temperature 2+ released from internal stores and the frequency of CASs. Another limitation is that we did not consider the volume of the extracellular space. Although the volume of astrocytes is not altered during CSD 2+ signals more accurately, the changes of volume of the extracellular space need be taken into account.Due to changes in the membrane potential are difficult to model because of the many and complex processes involved, we and others 2+ waves in astrocytes are thought to be transmitted by gap junction or by extracellular diffusion of ATP 3 through gap junction, and the ATP diffusion is not considered. Hence, the results in the present study are expected to be relevant to the brain structures in neocortex. To compare the properties of Ca2+ waves in different brain areas, the gap junction and ATP diffusion should be considered together. ATP-mediated Ca2+ waves have been modeled in the astrocyte network 2+ signals in astrocytes could be affected by the neuronal activity 2+ signals, especially the CSDCWs This model could be improved at least in two aspects. First, Ca2+ flows to the generation of CASs and CSDCWs, and indicate different mechanisms underlying CSDCWs and non-CSDCWs. An experiment test could be done is to example the effects of Ca2+ influx, especially Ca2+ influx via VGCCs, on the generation and propagation of CSDCWs. Research on the different types of Ca2+ signals might help to understand the different ways by which astrocytes participate in the brain functions.In summary, we analyze the contributions of different CaFigure S1The influence of CICR on CASs. From (A) to (E), the inhibition of CICR is 0%, 30%, 70%, 90% and 100%, respectively. By inhibiting CICR gradually, the frequency and amplitude of CASs decrease, while the duration increases.(TIF)Click here for additional data file.Figure S2The influence of SERCA on CASs. From (A) to (E), the inhibition of SERCA is 0%, 20%, 50%, 70% and 90%, respectively. By inhibiting SERCA gradually, the amplitude and duration of CASs decrease, while the frequency increases. Ca2+ dynamics evolve into small oscillations before disappearing.(TIF)Click here for additional data file.Figure S3The influence of VGCCs on CASs. From (A) to (E), the inhibition of VGCCs is 0%, 20%, 30%, 40% and 50%, respectively. In the present of CASs, inhibiting VGCCs has little influence on the amplitude and duration of CASs, but great on frequency.(TIF)Click here for additional data file.Figure S4Dynamics of CSD. During CSD, K+ in the ECS (A) and in the ICS (B) is significantly increased and astrocytes are depolarized at successive astrocytes in the network (C). The bar illustrates the time of locally elevating Ko to evoke a CSD.(TIF)Click here for additional data file.Figure S5The influence of temperature on the frequency of CASs. From (A) to (D), the value of temperature used in the model is 20, 25, 31, and 36°C, respectively. As the temperature increases, the frequency of CASs decreases. (E) CASs occur frequently at low temperature and become less frequent at higher temperature.(TIF)Click here for additional data file.Table S1The parameter values used in the model.(PDF)Click here for additional data file.Table S2The initial values of the variables.(PDF)Click here for additional data file.Materials S12+ flows through the membrane of ER and the dynamics of IP3 in single astrocytes in the previous model of CASs.Supporting materials that briefly describe the Ca(DOC)Click here for additional data file.


These findings are in line with the strong Ca2+-dependence of LB fusion activity and suggest that LRRK2 -/- affects exocytic response in ATII cells via modulating intracellular Ca2+ signaling. Post-fusion regulation of surfactant secretion was unaltered. Actin coating of fused vesicles and subsequent vesicle compression to promote surfactant expulsion were comparable in cells from LRRK2 -/- and wt animals. Surprisingly, surfactant (phospholipid) release from LRRK2 -/- cells was reduced following stimulation of LB exocytosis possibly due to impaired LB maturation and surfactant loading of LBs. In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca2+ signaling and promotes LB exocytosis upon stimulation of ATII cells with ATP.Leucine-rich repeat kinase 2 (LRRK2) is known to play a role in the pathogenesis of various diseases including Parkinson disease, morbus Crohn, leprosy and cancer. LRRK2 is suggested to be involved in a number of cell biological processes such as vesicular trafficking, transcription, autophagy and lysosomal pathways. Recent histological studies of lungs of LRRK2 knock-out (LRRK2 -/-) mice revealed significantly enlarged lamellar bodies (LBs) in alveolar type II (ATII) epithelial cells. LBs are large, lysosome-related storage organelles for pulmonary surfactant, which is released into the alveolar lumen upon LB exocytosis. In this study we used high-resolution, subcellular live-cell imaging assays to investigate whether similar morphological changes can be observed in primary ATII cells from LRRK2 -/- rats and whether such changes result in altered LB exocytosis. Similarly to the report in mice, ATII cells from LRRK2 -/- rats contained significantly enlarged LBs resulting in a >50% increase in LB volume. Stimulation of ATII cells with ATP elicited LB exocytosis in a significantly increased proportion of cells from LRRK2 -/- animals. LRRK2 -/- cells also displayed increased intracellular Ca LRRK2 is a ∼280 kDa protein with two enzymatic domains (Ras of complex GTPase domain and kinase domain) and several protein-protein interaction domains such as an amino terminal leucine-rich repeat domain and a carboxy terminal WD40 domain Despite the importance of LRRK2 for the pathogenesis in various diseases little is known about the cellular function of LRRK2. LRRK2 has been implicated in many different signaling pathways such as membrane trafficking 2+ concentration ([Ca2+]c) being at center stage 2+]c2+ influx via vesicular P2X4 receptors which promotes fusion pore expansion and subsequently facilitates surfactant release During LB exocytosis a sequence of highly regulated steps leads to fusion of exocytic vesicles with the plasma membrane, subsequent opening of a fusion pore and finally content release. Several intracellular signalling cascades stimulate LB fusion with the plasma membrane during the exocytic pre-fusion phase 2+ release from intracellular stores upon stimulation with ATP, which promotes triggering of LB exocytosis. However, using a biochemical approach to quantify surfactant secretion we found that surfactant release to the extracellular space was decreased after stimulation. This was not due to failure of actin coat formation and active extrusion of vesicle contents during the exocytic post fusion phase. In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca2+ signalling and promotes LB exocytosis upon stimulation of ATII cells with ATP.Within this study we investigated whether morphological changes of LBs observed in LRRK2 -/- mice are also present in LRRK2 -/- rats and whether they affect LB exocytosis and surfactant secretion. We measured single LB fusion events with the plasma membrane using high resolution, real-time fluorescent microscopy. ATII cells are particularly suitable for analysing single exocytotic events due to large size of secretory vesicles and slow, sequential vesicle fusion with plasma membrane. LB exocytosis has been intensively investigated in the past decade and there are several established microscopy methods for detection of single vesicle fusion events Rats were maintained at the central animal facility of the Ulm University according to institutional guidelines for ethical care of animals. All experiments in this study were approved by Regierungspräsidium Tübingen, grant Nr. 833.tm1sage, product number: TGRL4620) as well as age- and gender-matched wildtypes (Long-Evans). The age of animals ranged between 5 and 9 weeks.LRRK2 -/- rats were purchased from SAGE Labs , seeded on chamber slides , and used for experiments for up to 48 h after seeding.Alveolar type II cells were isolated from wt and LRRK2 -/- rats according to the procedure of Dobbs et al. Cells were washed twice in ice-cold DPBS  fixed for 20 min in 4% paraformaldehyde  in DPBS and permeabilised for 10 min with 0.2% saponin and 10% FBS  in DPBS. Cells were subsequently stained with primary (1∶300) and secondary (1∶400) antibodies in PBS, 0.2% saponin and 10% FBS for 30 min. Images were taken on an inverted confocal microscope  using a 63× lens (Leica HCX PL APO lambda blue 63.0×1.40 OIL UV). Images for the blue (DAPI), green (AlexaFluor 488), red (AlexaFluor 568) and far red (AlexaFluor 633) channel were taken in sequential mode using appropriate excitation and emission settings. All used primary antibodies were from Abcam  apart from antibodies against surfactant proteins B and C which were a gift from T. Haller . AlexaFluor phalloidin 568 and all conjugated secondary antibodies were from Invitrogen.2+]c and detection of individual LB fusion events was performed as described previously 2, 2 CaCl2, 5 glucose, 10 Hepes; pH 7.4) and kept in bath solution with 0.5 – 1 μM of FM 1–43. To efficiently induce LB exocytosis ATII cells were treated with various known and potent agonists for surfactant secretion: ATP (100 μM), PMA (300 nM), a combination of ATP and PMA, or ionomycin (1 μM) . The combined application of ATP and PMA was used because the previous report showed that the combined application can further increase LB fusion probability 2+]c as described in detail earlier The fura-2/FM1-43 assay for combined measurement of changes in the c = Kd×[(R−Rmin)/(Rmax−R)]×(Sf2/Sb2).We estimated the intracellular free Camin was measured after Ca2+ ionophore ionomycin (20 µM) was added to a Ca2+ free perfusion solution . Rmax was measured after ionomycin (20 µM) addition to Ca2+-containing perfusion solution . The proportionality coefficient Sf2 was measured as the maximal 380 nm fluorescence intensity after ionomycin addition to Ca2+-free solution. The second proportionality coefficient, Sb2, was measured as the minimal 380 nm fluorescence intensity after ionomycin addition to Ca2+-containing solution. We corrected both coefficients for the background fluorescence which was measured after MnCl2 quench  at the end of the experiment. We used 224 nM as a Kd value for fura-2 ATII cells were seeded in perfusion chambers  and stained with fura-2 AM (3 µM for 20 min). Ratio (R) was calculated from 340/380 nm excitation intensities in unstimulated cells in normal experimental bath solution. RATII cells were incubated with 100 nM LysoSensor Yellow/Blue (Invitrogen) at 37°C for 30 min. Cells were washed with experimental bath solution and imaged on the Zeiss Observer microscope  and 40× fluar oil objective using Metafluor software. After background subtraction, LB intensities at 340 and 380 nm excitation were measured in ImageJ and the 340/380 nm ratio was used for pH calculation.10((a−b)/(ratio−b)−1), where a, b and c are constants.Calibration of the ratio to pH relation was done as described previously We used Microsoft Excel and GraphPad Prism  for statistical analysis. Unless stated otherwise data are expressed as mean +/− SEM. n denotes true biological replicates  and non-paired student's t-test was used to assess the statistical differences between wt and LRRK2 -/- cell populations. Unless stated otherwise the number of experiments performed/cells analysed in each biological replicate was kept constant to account for correct weighting of individual biological replicates when generating means. Due to the low number of fusions/cell (on average 1–2 per responding cell) and within individual experiments (6–8 responding cells) fusion delay histograms were derived from pooled data from 3 to 6 animals. A non-parametric Mann-Whitney test was used to compare the medians of fusion delays after stimulation.The previous histological study of the lungs from LRRK2 -/- mice showed enlarged size and number of LBs in ATII cells Expression and localization of LB markers were not changed. LB membrane marker proteins Lamp1 and ABCa3 transporter were localized to the LB membrane in LRRK2 -/- as well as in wt cells  [38]. SuTo further investigate whether the observed increase in LB size is linked to changes in lamellar body homeostasis we also performed experiments to assess the pH in LBs from wt and LRRK2 -/- cells using the dye LysoSensor. No significant difference (p = 0.45) between the vesicular pH in LBs from wt animals  and LBs from LRRK2 -/- animals  could be observed. Approx. 150 to 250 LBs were analyzed per animal.3 mediated calcium release from internal calcium stores resulting in a “triggered” fusion response. PMA on the other hand acts via activation of protein kinase C without affecting intracellular Ca2+ concentrations and enables a more prolonged response to stimulation whereas ionomycin acts as an ionophore and causes massive Ca2+-entry from the extracellular space 2+ (ionomycin) signaling pathways and result in comparable activity responses  increased fusion response compared to wt cells   . When anctively) . These d2+]c using fura-2 while monitoring individual LB fusion events  in LRRK2 -/- cells and 0.49±0.19 µM (n = 2) in wt cells, respectively (p = 0.08) . However animals . Mean pe = 0.08) .Based on our finding that knock-out of LRRK2 results in a significantly enhanced LB fusion response in ATII cells when stimulated with 100 µM ATP, we next examined whether this also results in increased surfactant secretion. We analyzed the amount of secreted phospholipids (DPPC is the main component of surfactant) using a recently described enzymatic protocol To further elucidate these conflicting results – increased number of responding cells but reduced surfactant secretion in LRRK2 -/- cells – we investigated the potential impact of altered LRRK2 expression on the regulation of surfactant secretion during the post-fusion phase of LB exocytosis. The lipophilic nature of surfactant impedes rapid dispersal in aqueous solution and therefore surfactant does not readily diffuse out of fused LBs following opening of the exocytic fusion pore. We already showed that actin coating of fused LBs and subsequent actin coat compression play a pivotal role in surfactant expulsion from the vesicles Although LRRK2 was suggested to play a role in variety of diseases its cellular role remains elusive. Recent evidence implicated LRRK2 in regulation of secretory vesicle trafficking The original observation on increased LB size was made in LRRK2 -/- mice 2+]c2+ was described as a final trigger for LB fusion in ATII cells, the threshold necessary for fusion in ATII cells is very low (∼320 nM), and fusion kinetics correlate with [Ca2+]c kinetics 2+]c is not significantly elevated in LRRK2 -/- cells suggests that the increased rise in [Ca2+]c upon stimulation with ATP is the main effector of the increased fusion response in these cells. It is easily conceivable that in LRRK2 -/- cells the threshold for LB fusion is reached more readily and exceeded for prolonged times and hence more cells exhibit LB fusions upon stimulation with ATP. Such a model is also supported by the observation that in LRRK2 -/- cells the exocytic response following ATP treatment is similar to the response following stimulation that causes maximum elevation of [Ca2+]c and activation of Ca2+-dependent fusion activity (ionomycin). The increase in the rise of [Ca2+]c upon ATP treatment in LRRK2 -/- cells could be due to altered Ca2+ levels in intracellular Ca2+ stores or increased Ca2+ release kinetics. It is unlikely to result from altered Ca2+ extrusion mechanisms as the time-course of the [Ca2+]c decay is unchanged. Effects of LRRK2 on Ca2+ homeostasis and Ca2+ signaling have already been reported in other cell types 2+ homeostasis 2+ release from lysosomal Ca2+ stores 2+ release from intracellular stores. This difference could be due to the different signaling mechanism underlying Ca2+ mobilization in the different systems. In ATII cells ATP elicits Ca2+ release via activation of IP3 receptors 2+ mobilization 2+ release in LRRK2 -/- cells following stimulation with ATP likely facilitates or accelerates fusion of LBs that are already localized close to the plasma membrane 2+]c it barely affects recruitment or trafficking of additional LBs to the plasma membrane for fusion .Stimulation of primary ATII cells with 100 µM ATP resulted in a significantly increased percentage of cells that responded to stimulation with exocytic activity when LRRK2 expression was ablated. LB exocytosis is exceptionally sensitive to [Ca2+ independent LB fusion activity as PMA induced LB fusion response is unchanged in LRRK2 -/- cells. However, we cannot fully exclude the possibility that PMA already causes a maximum stimulatory effect that cannot be increased upon. It is well established that PMA causes a strong fusion response (i.e. more cells responding within a defined period of time after stimulation) due to the long lasting stimulatory effect. Hence the combined application of ATP and PMA, evoking a Ca2+ increase as well as direct stimulation of protein kinase C, does not necessarily need to exceed the PMA response LRRK2 -/- does not seem to affect Ca2+ signaling remains to be answered.The observed increase in exocytic activity in LRRK2 -/- cells is also in line with observations from cortical neurons where LRRK2 silencing increased fusion kinetics Shin et al showed that LRRK2 silencing in neurons leads to impaired compensatory endocytosis Although ATII cells from LRKK2 -/- rats contain larger LBs and these LBs had an increased propensity for fusion with plasma membrane upon physiological stimulation, the amount of secreted phospholipids following stimulation was lower than in wt cells. Therefore, we tested the possibility that the extrusion of secretory material might be impaired in LRKK2 -/- cells. Surfactant is a poorly soluble substance and the opening of the exocytic fusion pore is not sufficient for efficient surfactant release from the fused vesicle. Actin coating of fused LBs and myosin driven compression of this coat is necessary for active surfactant extrusion 2+ signaling and promotes LB exocytosis upon stimulation of ATII cells with ATP. However, further studies are required to fully elucidate the molecular mechanism how LRRK2 regulates intracellular Ca2+ release in these cells and whether LRRK2 affects surfactant loading of LBs. Results of these studies will also shed some more light on whether the observed effects of LRRK2 on exocytosis and secretion in other cell types are linked to changes in intracellular Ca2+ signaling.In summary our results suggest that LRRK2 -/- affects LB size, modulates intracellular Ca


At mucosal sites, papillomavirus genomes can persist in the epithelial basal layer following immune-mediated regression. Subsequent T-cell depletion stimulates a 3- to 5-log increase in the viral copy number, to levels associated with productive infection. Reappearance of microlesions was rare within the short time frame of our experiments but was observed in one instance. Our studies provide direct evidence that immunosuppression can trigger the reactivation of latent papillomavirus genomes, as previously proposed in humans. Definitive evidence of human papillomavirus (HPV) latency has been difficult to obtain, and for the most part has been limited to anecdotal reports of recurrence in individuals. The prevalence of HPV disease in immunosuppressed patients is thought to result from the reactivation of latent infections –8, and a–11–1–+ cells was apparent after 8 weeks of cyclosporine treatment compared to controls, there was no significant decrease in CD4+ cells, in accordance with the limited effect on the timing of regression that was seen. All of the rabbits showed adverse side effects upon cyclosporine treatment, including inappetence, lethargy, reduced weight gain, and abscess formation at the injection sites, as has previously been reported  rabbits, we next went on to apply this procedure to mature rabbits that had resolved the disease and had latent infections. Six rabbits were infected with ROPV prepared from rabbit warts, with one rabbit undergoing a mock infection in which the tongue was scarified but without the application of virions. At 4 weeks postinfection, rabbits were examined thoroughly under sedation to confirm that lesions had formed at all of the ROPV-inoculated sites  and C bun levels . Immunosn levels  and 26.1n levels  of thesen levels  and B ann levels  and B wen levels . Becausen levels . As we en levels  and F.The significant problems with intolerance of rabbits to cyclosporine were insurmountable in our experiments, and we were unable to reliably immunosuppress rabbits for longer than 12 weeks. Despite this difficulty, we went on to repeat the infection and immunosuppression experiments by infecting a further cohort of nine rabbits with ROPV and with induction of immunosuppression at 16 weeks postinfection in seven rabbits for a period of a further 16 weeks. In all infected rabbits, papillomas had formed and regressed as expected by 12 weeks postinfection, and both immunocompetent rabbits survived until the end of the experiment with no adverse effect of signs of papilloma recurrence. We were unable to sustain the immunosuppressed rabbits for the planned duration of the experiment, with all seven developing adverse clinical symptoms as a consequence of cyclosporine and dexamethasone administration  that necessitated culling on humane grounds. As a result, we were unable to examine the effects of immunosuppression more extensively in this additional group of animals. As an alternative, therefore, we decided to place infected tissues into an environment devoid of T cell immunity by grafting them under the kidney capsules of immunodeficient mice . By incuMus musculus has recently been described, and although MmuPV1 is likely to have distinct evolutionary adaptations that reflect its position as a Pi papillomavirus, it is anticipated that this model will be useful in further enhancing our understanding of latent infections (–On the basis of our previous studies, we proposed a model of papillomavirus latency in which viral gene expression is held in check in the epithelial basal layer by constant immunosurveillance and in which immunosuppression may be permissive of increased virus activity, as suggested in humans , 21, 22.fections –25.


In the present study we demonstrate that the Ca2+-activated, SK3 (KCa2.3) K+ channel, with high Ca2+-binding affinity, is also expressed in the mouse kidney . Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K+ secretion, the highest levels of expression were along the apical  cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca2+-dependent K+ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca2+-permeable TRPV4 channel, thereby inducing Ca2+ influx and elevating intracellular Ca2+ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca2+ influx, leading to elevated intracellular Ca2+ levels, activates this high Ca2+-affinity K+ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca2+-dependent regulation of membrane potential and K+ secretion.The Ca Ca, are a small group of potassium channels that are widely expressed in numerous tissues ranging from neurons to vascular endothelial cells + channels, the KCa channels can play a major role in regulating the plasma membrane electrical potential difference, Vm. However, their classical regulation by intracellular Ca2+, [Ca2+]i, leads to a highly dynamic coupling between Vm and [Ca2+]i which appears to underlie their central role in a wide array of functions ranging from neuronal excitability Ca channels expressed by a particular cell type, the hyperpolarization of the cell membrane following Ca2+-induced activation of a given KCa channel can either enhance Ca2+ influx through non-voltage-activated, Ca2+-permeable channels, such as TRP channels, or reduce Ca2+ influx in the case of voltage-activated Ca2+ channels Calcium-activated potassium channels, K2+-activated K+ channels have been identified: the large-conductance channel , the intermediate-conductance channel , and three small-conductance channels  2+. Further, the Ca2+ binding sites in the C-terminus, the “Ca2+ bowl,” of the channel-forming α-subunit of BK are characterized with a low Ca2+ binding affinity requiring high cytoplasmic levels of Ca2+ for activation  membrane of the late distal tubule and cortical collecting duct (CCD) are Ba2+-sensitive (blocker) channels that represent the dominant conductance of the apical membrane , an inward rectifier K+ channel from the Kir family, is the resting, Ba2+-sensitive, channel responsible for K+ secretion under normal physiological conditions + channels can contribute to K+ secretion. Indeed, it has been shown that elevated flow rates to the late distal tubule or the CCD leads to enhanced K+ secretion via activation of the luminal BK channel giving rise to the phenomenon of flow-dependent K+ secretion 2+-dependent process 2+ influx arising from activation of the Ca2+-permeable TRPV4 channel, a noted mechanotransducer channel + secretion or during other Ca2+-dependent K+-secretory stimulatory states is not known. Indeed, while a major fraction of the enhanced flow-induced K+ secretion is abolished in various animals models deficient in BK or certain regulatory β subunits + secretion under these conditions is currently not known. However, we have recently demonstrated that mouse M-1 collecting duct cells express both the BK channel and the SK3 channel where both channels are activated by mechanical stimulation in a Ca2+-dependent manner 2+ binding affinity of SK3, especially over BK, we speculate that the SK3 channel is an “early effector” that would respond to modest elevations in [Ca2+]i during K+ secretory stimulatory events where the channel would function as an important pathway contributing to regulation of Vm, K+ secretion and K+ homeostasis.In the mammalian kidney, Krane see . Hence, 2+ influx. We found that SK3 is expressed in the mouse kidney with immunohistochemical staining showing apparent strong expression in the thick ascending limb, the distal convoluted tubule, and the entire collecting duct systems, including the connecting tubule (CNT) and the cortical collecting duct (CCD). In the CCD SK3 was shown to be expressed in both principal cells (PC) and intercalated cells (IC), with pronounced expression along the apical  border and subapical regions. Selective activation of TRPV4, leading to Ca2+ influx, led to cell hyperpolarization that was partially inhibited by application of either apamin, a selective SK channel inhibitor, or iberiotoxin (IbTX), a selective BK channel inhibitor. These findings demonstrate that functional SK3 channels are expressed in the late distal tubule/collecting duct and that they are regulated by TRPV4-mediated Ca2+ influx where they would appear to play a key role in regulating membrane potential and K+ secretion in the TRPV4-positive cells of the CNT and CCD.The goal of this study was to determine if the SK3 channel was expressed in the late distal tubule and other nephron segments of the kidney and, if so, was it functionally regulated by CaC57BL/6 mice were maintained on a normal diet with free access to water. Kidneys were removed and used for experimentation as outlined for each protocol below. All studies were carried out in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the NIH. All animal protocols were approved by the Institute for Animal Care and Use Committee of The University of Texas Health Science Center (AWA#: A3414-01).5′-GCCCTGTTTGAAAAGAGAAAGCGAC-3′ and reverse: 5′-GCATCAGTGAAGAGTTTGCTATGGAGC-3′). SK3 primers were selected to cross the boundary been exon 2 and 3 to rule out products derived from genomic DNA. Nucleotide sequencing verified that the PCR product was derived from SK3 mRNA  against 100-bp standard markers (New England Biolabs).Total RNAs were prepared from whole kidney using TRIzol reagents (Invitrogen) following the manufacturer's instruction as described previously mRNA see . For BKαIn preparation for Western blotting, mice were anesthetized with isoflurane inhalation and the kidneys dissected free and immediately processed for immunoblotting. Briefly, kidneys were immediately sliced into several small pieces, on ice. Tissue was then homogenized with 4× volume of ice-cold lysis buffer , containing protease inhibitor cocktail . The homogenates were then immediately centrifuged at 15000 g for 15 min at 4°C and the supernatants collected and stored at −20°C until use.Ca2.3 directed against the N-terminus, 1∶100, Alomone Cat. #APC-025; see references For Western blots, 5× Laemmli buffer was added to sample protein and then heated for 10 min at 70°C. Next, 20 µg of sample protein was run in a 4–15% SDS-PAGE gradient gel, transferred to PVDF membrane, and blocked with 5% nonfat milk for 1.5 hrs at RT. Membranes were incubated with a well-characterized, high specificity, anti-SK3 primary antibody  overnight at 4°C see . Followi2 treatment and subjected to cervical dislocation. Subsequently, kidneys were dissected and cut into transverse sections (∼1 mm thick); then placed into ice cold PBS (pH 7.4). Medullary-cortical strips of tubules were dissected from the tissue and then individual CCD teased from the strips using watchmaker forceps (sites of bifurcation of the CCD were used to identify CCD segments from upstream connecting tubules). Isolated tubules were moved onto poly-L-lysine coated glass chips and placed in a perfusion chamber mounted on an Eclipse Ti Nikon microscope at room temperature. Tubules were then split-open with two sharpened micropipettes and used within 3 hrs of isolation for membrane potential measurements (see below).Sections of CCD were isolated and prepared from mouse kidney tissue as previously described 2(3) (Invitrogen), a voltage-sensitive fluorescent probe, was used to measure relative changes in cell membrane potential, Vm, of individual cells 2(3) dye has been widely used in a broad range of cells to report Vm. Unless otherwise noted, cells were bathed in a isotonic modified balanced salt solution (MBSS), containing (in mM): 140 NaCl, 5.4 KCl, 0.5 MgCl2, 0.4 MgSO4, 3.3 NaHCO3, 2 CaCl2, 10 Hepes, 5.5 glucose, and pH 7.4. Prior to imaging, cells were loaded with dye by incubation in MBSS containing 100 nM DiSBAC2(3) for 30 min at RT in the dark. The coverslips were mounted in a perfusion chamber (see above) on the stage of a high-resolution Nikon Eclipse Ti inverted fluorescence microscope equipped with a Lambda LS Xenon arc lamp illuminator and filter wheel (Sutter Instruments) and a CoolSNAP HQ2 cooled CCD camera (Photometrics) as before 2(3) was added to all perfusion solutions throughout the experiment. The fluorescence signal (images) was acquired using standard procedures (excitation wavelength = 530 nm and emission wavelength = 580 nm) + solution containing 50 mM KCl (High K+ solution: 50 mM KCl substituted for NaCl in MBSS) was used as a standard test for inducing a defined membrane depolarization. For statistical analysis, 7–15 ROIs from each split-open CCD were selected based on the Vm response to application of the High K+ solution. Typically from 3–5 CCDs were isolated and used from 2–4 kidneys (1–2 CCDs/kidney) for each treatment group. “n” is representative of the number of cells analyzed for all tubules in each group. Data are presented as a mean value ± SEM.DiSBACThe following chemicals were used in this study: GSK101  from a stock solution (1 µM) in DMSO; apamin  from a stock solution (1 mM) in PBS; and iberiotoxin  from a stock solution (0.1 mM) in PBS.t-test, when comparing only two groups, or a one-way ANOVA for larger groups followed by the Holm-Sidak a posteriori test to define significant differences among groups. The significance level was defined as P<0.05; n is the number of cells assessed in each group.Summary data are given as mean values ± SEM as indicated in the figures. Differences among groups was analyzed with either the As an initial step toward identifying the expression of SK3 channels in renal tubules, we assayed for both mRNA and protein expression levels using RT_PCR and immunoblotting from whole kidney samples. As shown in To identify the sites of expression of SK3 channels in renal tubules, mouse kidney sections (5 µm thick) were immunostained for SK3. We employed Alomone Lab's anti-SK3 antibody directed against the N-terminus as it has been shown to display high specificity for SK3 in a wide range of cell types and tissues see . Initial+/Ca2+ exchanger (NCX), a marker of the distal convoluted tubule and, as above, AQP2, a selective marker of PC cells of the collecting duct system  significant SK3 expression was apparent along the luminal cell border  with con+ secretion. The tubule segments in the upper portion of + secretion, it may be that SK3 contributes to this process under stimulated states.SK3 staining was also prominent along the distal convoluted tubule (DCT) segments, a site that is noted for ROMK expression and modest KKidney sections and isolated CCD tubules were used to identify the expression pattern of SK3 in the mouse collecting duct. Staining of kidney sections with SK3 and AQP2 revealed expression of SK3 channels along the entire collecting duct  and 5. F2(3), as done by others 2(3) reflects changes in Vm where an increase in fluorescence intensity  correlates with a depolarization of Vm and a decrease in fluorescence intensity with a hyperpolarization of Vm. A 30-min loading period with the dye (100 nM) provided an excellent fluorescence signal over background (2(3) fluorescent probe were also briefly exposed to a High K+ solution  or IbTX (50 nM) had little or no effect on Vm . Under basal [Cact on Vm  with RFUfore see , Vm typichannels . Indeed,2+-activated, small conductance, SK3 K+ channel in the nephron. Here we demonstrate for the first time that the SK3 protein is expressed in the mouse kidney with the highest levels of expression apparent in tubular segments of the distal nephron and collecting ducts. Using segment-specific markers we show that SK3 is expressed in the thick ascending limb, the distal convoluted tubule, the connecting tubule and the entire collecting duct system. Cellular sites of expression show dominant luminal membrane staining, although significant basolateral  membrane staining is apparent in some cells, especially in the earlier segments of the distal nephron. Since we also show that the channel is functional, being activated by Ca2+ influx and leading to hyperpolarization of Vm, it follows that SK3 may play a key role in Ca2+-dependent regulation of membrane potential and K+ transport in these distal nephron and collecting duct segments (see below). Indeed, the sites of SK3 expression largely mirror those for the Ca2+-independent ROMK channel , as done by many other groups 2+ influx, leading to membrane hyperpolarization as expected for activation of K+ channels, apamin now induced a significant depolarization of Vm demonstrating functional SK3 channels in the CCD following induction of TRPV4-mediated Ca2+ influx  or direct activation by addition of GSK101 2+ dependence of flow-induced K+ secretion in CCD is well known 2+ signaling + secretion by the CCD are markedly blunted 2+-dependent K+ channels and, in turn, Vm and K+ secretion in the CCD.Activation of SK3 in the mouse CCD by elevation of Cay in CCD . These f2+-activated K+ channels in CCD is still emerging, a similar close association between TRPV4 and SK3/IK channels has recently been shown in vascular endothelial cells. This association appears to be responsible, at least in part, for the “generation” of the endothelial-derived hyperpolarizing factor (EDHF) which is a key signaling factor leading to vascular vasodilations and a reduction in blood pressure (see 2+ influx will regulate activation of SK3 and IK and, in turn, Vm, a response similar to what we report here for SK3 and BK in the CCD.While the precise role of TRPV4 in control of Casure see . It has 2+ influx and regulation of Vm in the CCD. Nonetheless, it remains for future studies to define the nature of the association between TRPV4 and SK3/BK in the CCD and to identify potential microdomain and/or macromolecular structures that may give rise to the functional coupling among the channels.In other studies of vascular tissue it has recently been shown that TRPV4 and SK3 associate with each other in the endothelial cell plasma membrane + secretion and, hence, K+ homeostasis? Indeed, with the observed expression of SK3 at the apical cell membrane in distal nephron and collecting duct segments, it is likely that SK3 is a key contributor to K+ secretion under stimulated states that give rise to elevated cytosolic Ca2+ levels. It is well known that basal K+ secretion in the CNT and CCD, the main sites for K+ secretion, is attributable to the calcium-insensitive ROMK channels + secretion, such as with elevated fluid delivery to the distal tubule, the Ca2+-activated BK channel has been shown to be a key contributor to the enhanced K+ flux 2+ influx, which we and others have shown arises from flow-induced TRPV4 activation + secretion. While blockade of BK, or use of animals deficient in BK, has shown that a major fraction of the flow-induced K+ secretion is dependent upon BK, it has also recently been shown that the elevated flow rates stimulate ATP release from distal tubule cells + secretion + channel may be contributing to K+ secretion under these stimulated states. SK3 is postured to fit this role since the TRPV4-mediated Ca2+ influx will activate the channel in native CCD as shown in the current study. We also speculate that since SK3 has a much higher Ca2+ affinity than BK (see Does SK3 play a role in Kn BK see  that SK3+-channel dependent phenomena such as cell volume regulation. Cell swelling of many epithelial cells leads to enhanced Ca2+ influx and activation of Ca2+-activated K+ channels to induce K+ efflux, along with Cl−, leading to solute loss and regulatory volume decrease (see reviews 2+-sensitive apical K+ channels Ca channels. Others have shown that CCD cells in culture undergo regulatory volume decrease upon cell swelling 2+ influx in collecting duct cells 2+ influx which, in turn, leads to activation of both BK and SK3, it seems reasonable to conclude that swelling states in native CCD and CNT, at least for PCs, leads to activation of both BK and SK3 to effect cell volume regulation. However, the precise role of BK and SK3, or other Ca2+-activated K+ channels, in this process remains to be fully elucidated in future studies.Finally, SK3 is also likely to play a role in other K2+ levels and, as such, likely plays a key role, along with other Ca2+-activated K+ channels, in regulating both membrane potential and K+ secretion during states of elevated flow rates or cell volume regulation. It remains to be determined whether these Ca2+-activated K+ channels and Ca2+-permeable TRP channels function as independent entities or, more likely, associate into microdomains as macromolecular signalingplexes to bring about a coordinated control of channel functions in specific cell types of the distal nephron and collecting ducts.In summary, the current study provides evidence for the expression of functional SK3 channels along the mouse distal nephron with high levels of expression in the distal tubule and the entire collecting duct. In CCD, SK3 is expressed in both PCs and ICs, with prominent localization at the apical membrane and subapical regions of the cell. The SK3 channel is activated upon stimulation of TRPV4 and elevation of intracellular Ca


Traditional analyses of calcium homeostasis have separately quantified either calcium accumulation or release mechanisms. To define the system as a whole, however, requires multiple experimental techniques to examine both accumulation and release. Here we describe a technique that couples the simultaneous quantification of radio-labeled calcium accumulation in endoplasmic reticulum (ER) microsomes with the release of inorganic phosphate (Pi) by the hydrolytic activity of sarco-endoplasmic reticulum calcium ATPase (SERCA) all in the convenience of a 96-well format. After centrifuging the chilled homogenate at 10,000 × g for 15 minutes at 4°C, the resulting pellet containing nuclei, mitochondria and cellular debris was discarded. The supernatant was retained for subsequent ultracentrifugation at 100,000 × g for one hour at 4°C. The resultant supernatant was carefully decanted and discarded, and the pellet retained and washed twice with incomplete ice cold homogenization buffer (minus EDTA or protease inhibitors). The washed pellet was re-suspended in incomplete ice cold homogenization buffer and the protein concentration determined using the BCA method . Microsomes were diluted with the same buffer to a final protein concentration of 2.5 mg/ml and stored at -80°C in 1.2 mL aliquots.All animal procedures were approved by the Uniformed Services University of the Health Sciences Institution for Animal Care and Use Committee (IACUC) in accordance with international guidelines on the ethical use of animals. Whole brain microsomes were prepared as described previously . Briefly2 that was EGTA-chelated down to desired concentrations of free Ca2+. During assay development, it was found experimentally that a large, non-reactive bioreagent was required to maintain osmolar stability for assay function, for SR/ER preparations to reliably uptake as well as release Ca2+ over time in the presence of known classical second messengers. Without appropriate addition of polyethylene glycol (PEG), adding organelles with lumens, like ER, potentially burst from osmolar influx, even without activated Ca2+ uptake additions (ATP/Mg2+/Ca2+ for SERCA activity). In presence of uptake additions, highly variable, unpredictable 45Ca2+ accumulation occurs over time due to influx of osmolar gradients competing with Ca2+. Unless otherwise specified, all experiments were conducted in a buffer with 300 nM free Ca2+. Free Ca2+ concentrations were verified by a Ca2+ - EGTA calculator program [2+ concentration of 200 μM and required 361.6 mL of 10 mM EGTA to titrate the free Ca2+ concentration to the desired concentration of 300 nM. Prior to beginning the experiment, additional fresh reagents were prepared and added to 1.2 mL of assay buffer. This pre-mix includes: 2 mM adenosine 5' triphosphate (ATP), 2 mM MgCl2, 1,4 dithiothreitol (DTT), 5 mM phosphocreatine (PCr), and 20 U/mL creatine phosphokinase (CPK). SERCA activity is Mg2+- and ATP-dependent, and for every two Ca2+ ions transported, hydrolyzes one ATP to ADP. The CPK and PCr serve as a buffer ATP-regeneration system replenishing ATP stores to maintain SERCA activity under experimental conditions [45Ca2+ was lastly added to the assay buffer at a final concentration of 0.2 μCi/mL.This assay was developed in our lab to run in a 96-well microplate format, using a 0.45 μm glass fiber type B MAFB micro filter plate from Millipore  to allow data collection from both the microsomes and the filtrate. The uptake reaction was run in assay buffer prepared to our specifications by World Precision Instruments  that contained 20 mM HEPES, pH adjusted to 7.35 with KOH, 80 mM potassium chloride (KCl), 3% (w/v) polyethylene glycol , 5 mM sodium azide , 25 mM potassium oxalate, and 200 μM CaCl program . In our nditions ,20. Radi2+ accumulation for a total of 180 minutes in 30 minute intervals and did not see a decrease in the reaction slope until after 90 minutes (data not shown). The plastic filtrate capture basin normally used was replaced by a clear microplate placed in the vacuum plate base immediately beneath the filter plate such that all cells were aligned to capture the filtrate. Because of their size and charge, microsomes with accumulated luminal 45Ca2+ remained trapped in the microplate wells [2, and 2 mM EGTA to remove non-specific signal. Finally, 50 μL of Ready Value scintillation fluid  was added to each well of the filter plate and the radioactive signal was measured in a Wallac microbeta liquid scintillation counter  to determine 45Ca2+ accumulation. Non-specific uptake was considered 45Ca2+ accumulation in the presence of 10 μM A23187, a potent and highly selective Ca2+ ionophore.To start the experiment, a 96-well plate was loaded with assay buffer , microsomes (100 μg/mL), test reagents , and the pre-mix. The covered microplate was incubated at 37°C for 60 minutes, and the reaction terminated by filtration using a Millipore vacuum plate base (MAVM0960R). We previously determined the rate of accumulation was still in the linear phase at 60 minutes. We measured microsomal Cate wells . Filtersi that complexes with ammonium molybdate and malachite green following release from SERCA-mediated ATP hydrolysis [i contamination. Briefly, the reagent to quantify Pi was prepared by mixing 1 volume of 10% (w/v) ammonium molybdate in 4 M HCl with 3 volumes 0.2% (w/v) malachite green in 4 M HCl, followed by stirring for 30 min then gravity filtration. The initial 20% of the filtrate was discarded due to dilution by the HCL rinse. Inorganic phosphate reagent was stored in the dark for no more than two weeks before replacement. Experiments were conducted to optimize parameters such as time, enzyme concentration, and reagent concentrations,(data not shown). When Pi is complexed with ammonium molybdate and malachite green in 4 M HCl, it creates a green color which can be quantified by reading the absorbance spectrophotometrically at 660 nm in a FLUOstar Omega  and compared to a standard curve of known Pi concentrations [SERCA activity was measured using a colorimetric assay that quantifies the amount of Pdrolysis . All glatrations .4 in assay buffer which was stored at -20°C when not in use. This stock solution was then further diluted in assay buffer to generate a standard curve ranging from 0 to 15 nmols of PO42-, each in a final volume of 200 μL. Fifty μL of malachite green/ammonium molybdate dye reagent was added to each sample and color allowed to develop for 10 minutes at room temperature before being quantified by absorbance spectrophotometric analysis at 660 nm in a FLUOstar Omega . To quantify Pi in the filtrate, a 15 μL aliquot was removed and combined in a fresh microplate containing 185 μL assay buffer and 50 μL dye reagent. Once again, the colorimetric reaction was allowed to proceed for 10 minutes before the OD660 was measured spectrophotometrically.A standard curve was generated by preparing a stock of 10 mM NaHPO2+ radiotracer accumulation in ER microsomes with the hydrolysis and release of Pi by ER resident Ca2+-ATPases (SERCAs). SERCA activity is highly dependent on the availability of both Mg2+ and ATP [45Ca2+ accumulation in microsomes on increasing concentrations of Mg2+. In this experiment, ATP concentration was held constant at 2 mM, while Mg2+ concentration was increased from 0 to 10 mM. Calcium accumulation was measured without (control) or with 1 μM TG. A clear dependence on Mg2+ was observed with activity not rising above background levels until 100 μM of Mg2+ was present. A plateau in activity was obtained when Mg2+ levels reached 3-5 mM. As expected, Mg2+ concentrations above 5 mM competitively inhibited Ca2+ binding sites on the SERCA, thus reducing Ca2+ accumulation in the microsomes [45Ca2+ accumulation [2+ to maximize Ca2+ accumulation, 3 mM Mg2+ was used for all subsequent experiments.These experiments establish a more robust technique coupling quantification of Ca and ATP . Figure crosomes ,24. Alsomulation ,25. Havi2+ accumulation assays were repeated using a constant concentration of Mg2+ (3 mM). As with the low levels of Mg2+ in Figure 2+ accumulation was not detectable at ATP concentrations below 50 μM. VMax was reached at 2.5 mM ATP. Higher concentrations of ATP stimulate the opening of IP3 release channels thereby reducing Ca2+ accumulation in ER microsomes [45Ca2+ accumulation was TG-sensitive.To determine the best ATP concentrations Figure , Ca2+ accrosomes . In addi2+ concentration, and chosen a physiologically relevant ATP level (2.0 mM), we investigated the pharmacokinetics of SERCA inhibition on ER Ca2+ accumulation and Pi release under the same assay conditions, with data collected simultaneously. TG, experimentally the most potent and commonly used specific inhibitor of SERCA [2+ accumulation in ER microsomes  [2+ accumulation until doses in excess of 100 μM are reached, representing the TG-resistant (TG-R) pool (data not shown). The TG-R pool represented approximately 20% of the total sequestered Ca2+ in our assays.Having optimized the Mgof SERCA ,26, signs Figure . At low 2+ accumulation, the measurement of Ca2+ accumulation was directly coupled to the simultaneous quantification of Pi release  of TG. Under control conditions, both Ca2+ uptake and Pi release are significantly higher (p < 0.05) than seen in the presence of TG (1 μM). The sample data collected from assays shown in Figure 2+ accumulation and Pi release in microsomes for three separate conditions: control , and addition of either 100 nM TG or 10 μM A23187. In this example, the control reaction resulted in the largest amount of 45Ca2+ accumulated in the ER microsomal preparation as measured by liquid scintillation counting with 10,311 ± 700 counts per minute (CPM), corresponding to an activity of 7.10 nmol 45Ca2+/min/mg prot. As expected, the amount of 45Ca2+ accumulated in ER microsomes from the same preparation containing TG, was reduced by 70% with only 3142 ± 345 CPMs (2.16 nmol 45Ca2+//min/mg protein). The difference in 45Ca2+ uptake between control and TG represented the TG-S SERCA activity and agreed with previous reports [2+ ionophore A23187. This pharmacological agent creates numerous Ca2+ permeable pores in biological membranes and thus limits the accumulation of Ca2+ within microsomes. Consequently net accumulation of 45Ca2+ in the presence of A23187 was by far the lowest of the three treatment groups with only 1064 ± 74 CPMs (0.73 nmol 45Ca2+/min/mg prot). Pi released , followed by the control sample with an A660 of 0.551(756 nmol/min/mg prot), and finally the TG treated sample with an A660 of 0.484 (654 nmol/min/mg prot).Having optimized the assay conditions for Cae Figure . The fir reports ,27. The d Figure  from ATP2+ accumulation in microsomes and the release of Pi from hydrolysis of ATP by SERCA in a 96-well format under the same experimental conditions. Calcium-mediated processes are ubiquitous throughout the cell and must be properly maintained as any significant disruption in Ca2+ regulation will impair cellular function and may lead to cell death [2+ and use it as a second messenger. The technique described herein provides a robust tool developed from coupling two fundamental assays for investigating the effects of various metabolites, reagents, ligands, and substrates on the mechanisms of Ca2+ sequestration within the ER.The key result of this study was the successful quantification of both Call death . Consequ2+ accumulation simultaneous with release of inorganic phosphate is a direct correlation of two linked parameters, increasing the power of data interpretation far beyond the capability from performing either assay alone. A change in ER Ca2+ accumulation over time is the sum of two major processes: Ca2+ accumulation, predominantly by ATP-dependent SERCA, and Ca2+ release or loss through several different mechanisms, such as resident IP3 and ryanodine receptor channels, or passive leak pathways. Quantification of SERCA-dependent ATP hydrolysis provides a direct measure of SERCA activity. By then comparing the net Ca2+ accumulation to SERCA activity, the amount of Ca2+ lost via release mechanisms and leak pathways can then be determined mathematically. This technique significantly expands upon the method introduced by Karon et al., who described a continuous spectrophotometric method to simultaneously measure changes in free Ca2+ concentration and ATPase activity [The specific advantage of assaying Caactivity . Howeveri quantification is extremely sensitive, capable of reliably detecting Pi at concentrations as low as 100 pM [i from a sample may be required prior to analysis if there is a low signal:noise ratio or minimal treatment effect. Endogenous Pi is typically removed by desalting soluble protein fractions using low speed centrifugation through small columns of Sephadex G50 (300 μl of supernatant per 3 ml of G50) equilibrated in incomplete microsome homogenization buffer to remove endogenous ions, small molecules and free phosphate. Additionally, assay conditions may have to be changed depending on the specific application. For example, in Figures 2+ uptake. In these experiments, phosphocreatine (PCr) and creatine phosphokinase (CPK) were included to regenerate ATP and maintain a constant concentration prior to experimental endpoint. Since PCr and CPK increase the Pi background, they can be omitted for assays not requiring maximal Ca2+ accumulation. Therefore, no ATP regenerating system was included in Figure The ammonium molybdate/malachite green technique of Ps 100 pM . Due to i, including the plasma membrane ATPases (PMCAs), the Golgi apparatus secretory protein ATPases (SPCAs) and so on. This fact, along with exquisite assay sensitivity, helps explain why Pi measurements do not follow strict stoichiometric rules. The fact that TG potently inhibits SERCA allows us to exploit this tool experimentally and account for these other sources of Pi. Pi generated from SERCA activity is defined by the difference in Pi generated in one condition minus the Pi generated in the same condition in the presence of TG. The concentration of TG needed to inhibit PMCA or SPCA is 10 to 300 times greater than that necessary to inhibit SERCA [There are many processes that may contribute to the endogenous generation of Pit SERCA .45Ca2+ . Maximal accumulation of Ca2+ is the net difference between SERCA dependent influx and efflux through Ca2+ release channels and passive leak pathways. The amount of Pi released from ATP hydrolysis directly measures SERCA activity and was used to distinguish the relative contribution of SERCA activity compared to efflux mechanisms on the accumulation of Ca2+. These data represent baseline experimental conditions for comparison to subsequent treatments. As expected, the addition of TG (TG) significantly reduced microsomal Ca2+ accumulation. TG irreversibly inhibits the formation of E-P intermediates of P-type ATPases such as SERCAs, thus reducing the influx of Ca2+ by the preventing SERCA conformational changes and ATP hydrolysis [i was reduced in the presence of TG  to measure the effect of mutations in presenilin 1 (PS1) on the concentration of free Ca2+ within the ER lumen [2+ load despite having a higher rate of ER Ca2+ filling and vice versa. The only way to accurately determine the effect of the mutation of interest (or compound of interest) on the rate of SERCA is to measure the activity directly using a technique such as SERCA mediated ATPase activity and not to infer it by measuring a parameter that is the sum of many moving parts. The technique described herein will provide a valuable tool that will compliment the newer state-of-the-art Ca2+ measuring techniques. Researchers aiming to quantify the effect of their compound of interest on SERCA activity and Ca2+ release mechanisms can follow up their initial observations of changes in Ca2+ homeostasis with quantification of the SERCA activity.In recent years, the CaAequorin . These pAequorin . These tER lumen . They id2+ accumulation within microsomes provides a much more detailed understanding not only of the relative contribution of SERCA activity, but also by pharmacological manipulation and deductive analysis, the role of both active release and passive leak pathways. This technique can be easily modified to analyze not just ER microsomes, but also cell preparations and tissue homogenates, as well as a range of substrates, metabolites and pharmacological agents.The coupling of two well-established techniques to analyze mechanisms of CaThe authors declare that they have no competing interests.DCM conducted the experiments, assisted with data interpretation, and assisted with manuscript preparation. WSK assisted with technical preparation. AV, JTC, and WDW assisted with data interpretation and manuscript preparation. All authors read and approved the final manuscript.The opinions expressed herein belong solely to the authors. They do not nor should they be interpreted as representative of or endorsed by the Uniformed Services University of the Health Sciences, U.S. Army, U.S. Navy, Dept. of Defense or any other agency of the federal government.Step by step procedure.Click here for file


Cajanus cajan (L.) Millsp. The present study explores the underlying cellular mechanisms for CSA-induced relaxation in rat renal arteries. Vascular reactivity was examined in arterial rings that were suspended in a Multi Myograph System and the expression of signaling proteins was assessed by Western blotting method. CSA (0.1–10 µM) produced relaxations in rings pre-contracted by phenylephrine, serotonin, 9, 11-dideoxy-9α, 11α-epoxymethanoprostaglandin F2α (U46619), and 60 mM KCl. CSA-induced relaxations did not show difference between genders and were unaffected by endothelium denudation, nor by treatment with NG-nitro-L-arginine methyl ester, indomethacin, ICI-182780, tetraethylammonium ion, BaCl2, glibenclamide, 4-aminopyridine or propranolol. CSA reduced contraction induced by CaCl2 (0.01–5 mM) in Ca2+-free 60 mM KCl solution and by 30 nM (−)-Bay K8644 in 15 mM KCl solution. CSA inhibited 60 mM KCl-induced Ca2+ influx in smooth muscle of renal arteries. In addition, CSA inhibited contraction evoked by phorbol 12-myristate 13-acetate  in Ca2+-free Krebs solution. Moreover, CSA reduced the U46619- and PMA-induced phosphorylation of myosin light chain (MLC) at Ser19 and myosin phosphatase target subunit 1 (MYPT1) at Thr853 which was associated with vasoconstriction. CSA also lowered the phosphorylation of protein kinase C (PKCδ) at Thr505. In summary, the present results suggest that CSA relaxes renal arteries in vitro via multiple cellular mechanisms involving partial inhibition of calcium entry via nifedipine-sensitive calcium channels, protein kinase C and Rho kinase.Cajaninstilbene acid (CSA) is a major active component present in the leaves of  Cajanus cajan (L.) Millsp (pigeon pea) in vitroCajaninstilbene acid CSA, , one of The pharmacological activity of CSA in the vascular system is unknown. It is possible that CSA also benefits vascular function. Therefore, the present study was designed to examine the cellular mechanisms for CSA-induced relaxation and roles of signaling molecules involved in the regulation of contractility in rat arteries.CSA produced concentration-dependent relaxations to similar degrees in isolated renal arteries pre-contracted with 60 mM KCl, phenylephrine, serotonin and U46619 , Table 1CSA-induced relaxations showed no gender difference since they were comparable in both male and female rat arteries . Treatme2 (inwardly rectifying potassium channel blocker at 10 µM), glibenclamide (ATP-sensitive potassium channel blocker at 10 µM), TEA+ , 4-AP (voltage-sensitive potassium channel blocker at 1 mM) and propranolol (non-selective β-adrenoceptor antagonist at 1 µM) (CSA-induced relaxations were unaffected by 30-min exposure to BaClat 1 µM) .2+ influx in CSA-induced relaxation, the arteries were incubated in a Ca2+-free depolarizing solution containing 60 mM KCl. The representative trace in 2 into this bathing solution caused concentration-dependent contractions. Thirty-minute treatment with CSA (0.1–10 µM) inhibited CaCl2-evoked contraction while nifedipine  was used as positive control  in a Casolution .2+-free solution for 60 min, the levels of phosphorylation of MLC at Ser19  partly through inhibition of Rho kinase and PKC-dependent contractile mechanisms in vascular smooth muscle cells.The present study examined the vascular reactivity of cajaninstilbene acid in rat renal arteries and provided novel findings regarding its pharmacological properties. CSA produced endothelium-independent relaxation (1) partly through antagonism of CaPrevious studies showed that CSA possesses an estrogen-like activity on osteoblast and osteoclast The endothelium regulates vascular tone, while hypertension and atherosclerosis are associated with the impaired endothelial function; the latter is usually caused by disturbed balance in endothelium-derived relaxing and contracting factors + channels, thus discounting the involvement of K+ channel. Elevated KCl is known to activate voltage-gated Ca2+ channels via membrane depolarization in VSMCs. Therefore, high KCl-induced contraction was reduced by CSA, suggesting that CSA is likely to interfere with Ca2+ influx via Ca2+ channels. This notion was further supported by the following three observations. First, CSA progressively inhibited Ca2+-triggered contraction in a Ca2+-free, 60 mM KCl-containing Krebs solution. Second, CSA also concentration-dependently reduced contractions evoked by the Ca2+ channel activator (−)-Bay K8644 with similar potency as in KCl-evoked contraction. Lastly, CSA prevented the rise of [Ca2+]i in smooth muscle cells in situ of renal arteries upon the addition of 60 mM KCl.CSA-induced relaxations were not affected by elevated KCl nor by individual blockers for various types of K2+ rise 2+-free Krebs solution, suggesting that PKC inhibition may be involved. The downstream targets of both calcium-dependent and -independent mechanisms mediating vascular smooth muscle contraction are phosphorylation of myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1) The present results also suggest that in addition to calcium antagonism other cellular mechanisms may contribute to CSA-induced relaxations as CSA is more effective in relaxing arteries contracted by receptor agonists than by elevated KCl. Constrictive agonists used in this study can activate MLCK, PKC and Rho kinase; the latter two are independent of intracellular Cain vitro likely through both antagonism of calcium entry via nifedipine-sensitive Ca2+ channel and inhibition of cellular pathways in association with PKC and Rho/Rho kinase. The present results indicate that CSA and CSA-containing herbs can be of potential benefits in reducing the elevated VSMC tension which is implicated in cardiovascular pathogenesis although such benefit needs in-depth investigation in animal models of hypertension and other vascular pathologies.In summary, the present study provides novel evidence showing that CSA relaxes renal arteries 2, 2.5 CaCl2, 25 NaHCO3, 1.2 KH2PO4 and 11 D-glucose.The investigation conforms to the Guidelines for the Care and Use of laboratory animals published by the National Institutes of Health. Both male and female Sprague-Dawley rats (250∼300 g) were killed by cervical dislocation. The kidneys were removed and dissected in ice-cold oxygenated Krebs solution. Krebs solution contained the following composition (in mM): 119 NaCl, 4.7 KCl, 1 MgCl2–5% CO2 and maintained at 37°C at pH ∼7.4 as described previously Rings (∼2 mm in length) of renal arteries were isolated from adhering connective tissues. Each segment was mounted in a Multi Myograph System , bathed in Krebs solution bubbled with 95% OPhenylephrine (0.5 µM), U46619 (100 nM), serotonin (1 µM) and KCl (60 mM) were used to induce steady contraction tone in endothelium-intact rings, concentration-response curves were subsequently studied by cumulative addition of cajaninstilbene acid . The time-matched vehicle (DMSO) control protocol was also performed.+ (3 mM), BaCl2 (10 µM), glibenclamide (10 µM), 4-AP (1 mM) or propranolol (1 µM).In the second set of experiments, CSA-induced relaxation was obtained in endothelium-intact and endothelium-denuded rings. Some rings with intact endothelium were exposed for 30 min to L-NAME  2+ influx through L-type Ca2+ channels, CaCl2 (0.01–5 mM) was added in Ca2+-free, 60 mM KCl solution containing 100 µM EGTA after pre-incubation of CSA  or nifedipine  for 30 min. In addition, some rings were pre-contracted by (−)-Bay K8644  in 15 mM KCl Krebs solution To determine the effect of CSA involved in Ca2+-free Krebs solution containing 100 µM EGTA and then relaxed by CSA.Finally, the calcium-independent mechanisms were studied in endothelium-denuded rings. Each ring was contracted by phorbol 12-myristate 13-acetate (PMA at 10 µM) in a Ca2 and 5% CO2 and 10 mM BDM  at 37°C. Using an Olympus Fluoview FV1000 laser scanning confocal system (Olympus), fluorescence was measured continuously every 20 s (excitation: 495 nm and emission: 505–525 nm). The fluorescence intensity at a certain time point (F1) was compared to that at the starting point of image recording (F0) to show the changes of intracellular Ca2+ concentration [Ca2+]i upon the addition of 60 mM KCl.Renal arteries were dissected free from connective tissue and cut open longitudinally. The vascular strips were incubated with 5 µM calcium indicator fluo-4 AM (Molecular Probes) in Krebs solution for 1 h at 37°C as described before 2+-free Krebs solution containing 100 µM EGTA with or without 10 µM CSA for 60 min or incubation with 30 nM U46619 with or without 10 µM CSA for 30 min in Krebs solution, renal arteries were frozen in liquid nitrogen and homogenized in ice-cold RIPA lysis buffer containing 1 µg/ml leupeptin, 5 µg/ml aprotonin, 100 µg/ml PMSF, 1 mM sodium orthovanadate, 1 mM EGTA, 1 mM EDTA, 1 mM NaF, and 2 mg/ml β-glycerolphosphate as described previously After incubation with 10 µM PMA in CaCajamus cajan (L.) Millsp. Phenylephrine, acetylcholine, serotonin, L-NAME, indomethacin, TEA+, ICI-182780, glibenclamide, BaCl2, 4-AP, propranolol, nifedipine, (−)-Bay K8644, and PMA were from Sigma and U46619 was from Cayman. Indomethacin, ICI-182780, glibenclamide, nifedipine, (−)-BayK 8644, PMA, U46619 and CSA were dissolved in DMSO and others in double-distilled water.Cajaninstilbene acid (purity≥99%) was isolated from 2 refers to the negative logarithm of the dilator concentration that caused half of the maximal relaxation and Emax% refers to the maximum relaxation. Concentration-response curves were analyzed via GraphPad software (Version 4.03) and statistical significance was compared by two-tailed Student’s t-test or one-way analysis of variance followed by Newman-Keuls test. Values of p<0.05 was considered statistically significant.Data are means±SEM of n rats. pD


Residual Ca2+ is sensed by Ca2+-binding proteins, among other potential actions, it mediates time- and space-dependent synaptic facilitation and depression via effects on CaV2 channel gating and vesicle replenishment in the readily releasable pool (RRP). Calmodulin, a Ca2+-sensor protein with an EF-hand motif that binds Ca2+, interacts with CaV2 channels and autoreceptors in modulation of SNARE-mediated exocytosis.Synaptic efficacy is remodeled by neuronal firing activity at the presynaptic terminal. Presynaptic activity-dependent changes in transmitter release induce postsynaptic plasticity, including morphological change in spine, gene transcription, and protein synthesis and trafficking. The presynaptic transmitter release is triggered and regulated by Ca Presynaptic firing of action potentials activates voltage-gated Ca2+  (CaV) channels, and Ca2+ entry initiates release of neurotransmitters. Ca2+  dependence on fast neurotransmitter release is thought to be conferred by the synaptotagmin, a family of Ca2+ sensors that interact with SNAREs [2+ and ensure the synchronization of Ca2+-dependent exocytosis with the presynaptic action potential [2+-binding proteins sensing residual Ca2+, which accumulates locally in the presynaptic terminal during trains of action potentials, may act as potential effectors for these reactions. Considerable evidence supports a role for calmodulin (CaM), another family of Ca2+ sensors with an EF hand motif that binds Ca2+, in modulation of SNARE-mediated exocytosis [2+ channels [2+/ CaM kinase II [2+-binding protein with an EF hand motif, parvalbumin, acts as a mobile presynaptic Ca2+ buffer that accelerates withdrawal of residual Ca2+ and decay of short-term facilitation in the calyx of held [2+ effector or Ca2+ buffer to regulate transmitter release probability [For memory formation in a neuronal circuit, the primary function of presynaptic terminals is the firing activity-dependent release of neurotransmitters and subsequent recycling of their carrier synaptic vesicles, processes which critically depend on ATP and Cah SNAREs . Synaptootential . Neuronaocytosis , 7 and eocytosis , 9. Targchannels , Ca2+/ Cinase II , rab3 [1inase II ), and eninase II ). Anothe of held  and GABA of held . Calretibability . via acute microinjection of cDNA, small interfering RNA (siRNA), dominant-negative transgenes, peptides, antibodies, and metabolites [2+ effector sensing residual Ca2+ that mediates time- and space-dependent synaptic depression and facilitation via effects on CaV2 channel gating [A large number of proteins are involved in presynaptic function; their synthesis, transport, and function appear to be regulated by presynaptic firing activity –20, Sympabolites , 24–27, abolites , 28. Ourl gating  and vesil gating .V2.1 channels are densely clustered [2+  entered through CaV2.1 channels initiates synaptic transmission [V2.1 channels are Ca2+-dependently regulated by CaM [2+-binding proteins, calcium-binding protein 1 and visinin-like protein-2 [2+-binding proteins interact with a bipartite regulatory site in the intracellular C terminus of the α12.1 subunit [α12.1IM-AA) blocked Ca2+-dependent facilitation of CaV2.1 channels [2+-dependent inactivation was blocked in channels lacking the adjacent CaM-binding domain  [At most fast synapses in the central nervous system, Calustered , and Ca2smission , 32. CaVd by CaM , 33–36 arotein-2 –39. CaM channels , 36, whe2.1ΔCBD) , 39. 2+/CaM-dependent inactivation of CaV2.1 channels is dependent on global elevations of Ca2+ [2+ channels, local Ca2+ buffers, the volume of the intracellular compartment, and other differences in the cellular context in which the channels are located. Ca2+-dependent inactivation of CaV2.1 channels is observed in transfected cells overexpressing CaV2.1 channels [V2.1 channels are densely localized and large Ca2+ transients are generated. In contrast, Ca2+-dependent inactivation is not reliably observed in the large neuronal cell bodies of Purkinje neurons [Ca of Ca2+ , which ichannels , 34, 35 channels , 41, whe neurons  or SCG n neurons .2+/CaM-dependent regulation of CaV2.1 channels [α12.1WT. PPD with short interstimulus interval (<40 ms) was blocked by the α12.1ΔCBD transfection, while PPF with intermediate interstimulus interval (50–100 ms) was blocked by the α12.1IM-AAtransfection. Thus, temporal regulation of CaV2.1 channels contributes to PPD and PPF by changing the release probability in an activity-dependent manner in response to Ca2+ entry during the first action potential of the pair, suggesting a spatial change in Ca2+ concentration at the active zone and also regulates CaV2.1 channel activity.Paired-pulse facilitation (PPF) and paired-pulse depression (PPD) are both mediated by Cachannels . EPSPs rin vivo. During bursts of action potentials, the CaM-dependent regulation of CaV2.1 is involved in the short-term synaptic plasticity , last for seconds to minutes . In SCG iseconds . In contmulation  and alsomulation , 50.2+ are temporally sensed by a Ca2+ sensor CaM, resulting in Ca2+-dependent facilitation and inactivation of presynaptic CaV2.1 currents. These currents can mediate short-term synaptic facilitation and depression, which are conserved forms of plasticity at many different types of synapses [V2.1 channels, residual Ca2+ can actually control “instantaneous” Ca2+ during an action potential, shape the local Ca2+ transient at the active zone, and cause facilitation, augmentation, and depression of synaptic transmission. The postsynaptic response is, therefore, controlled by Ca2+/CaM-dependent modulation of presynaptic Ca2+  entry, which acts to encode the information contained in the frequency of presynaptic firing of action potentials for transmission to the postsynaptic cell. Thus, presynaptic Ca2+ channel regulation by CaM is a major molecular mechanism underlying information processing in the nervous system.Short-term synaptic plasticity is crucial for encoding information in the nervous system . Changessynapses . Evident2+-dependent CaM binds to the intracellular C-terminal tail (ct) of group III mGluRs [2+ [2+ level for the expression of short-term facilitation, a molecule that can bind to the ct region of group III mGluRs and have an interaction with the receptor that is regulated by Ca2+-activated CaM is a good candidate for mGluR-mediated expression of short-term synaptic facilitation.Presynaptic group III metabotropic glutamate receptors  are expressed in the presynaptic active zone and reduce synaptic vesicle release upon stimulation by agonists –55. A prI mGluRs , 57. BecluRs [2+  correspoin vitro binding experiments indicated that either mGluR4 or mGluR7 directly interacted with Munc18-1 in the brain. Group II mGluRs are also expressed presynaptically and modulate synaptic transmission [Munc18-1 is a protein essential for neurotransmission  and promsmission –55. Howesmission ; it bindsmission . 2+-activated CaM at a concentration of residual Ca2+ [2+-binding motif, the interaction between ct-mGluR4 and Munc18-1 is enhanced with increasing concentrations of Ca2+, with saturation at 3 μM. In the presence of CaM, mGluR4 binds Munc18-1 at the resting Ca2+ level of 0.1 μM and releases it above the Ca2+ level of 1 μM, corresponding to the residual Ca2+ level after an action potential.CaM binds to the membrane-proximal region of group III ct-mGluRs , 62. Munual Ca2+ . AlthougFunction of the mGluR4/Munc18-1 interaction in synaptic transmission is the reduction of basal transmitter release . At syna2+-sensing mechanism, mGluR4C appears to be responsible for the expression of short-term synaptic facilitation; at the very least it reduces short-term synaptic depression. Acutely, PPD, but not PPF, was suppressed in the presence of mGluR4C peptide, suggesting that mGluR4 peptide introduces a facilitating property to these synapses which is apparent when these synapses exhibit PPD. This evidence is consistent with the idea that the absence in the ct region of mGluR4 accounts for the impaired expression of PPF in mGluR4-KO mice [Generally, a decrease in the initial probability of transmitter release leads to a larger synaptic enhancement or a reduction of synaptic depression , 64. Fro-KO mice .2+ activates CaM, which in turn liberates Munc18-1 from mGluR4, causing short-term synaptic facilitation je cells . If Munc2+-binding proteins that sense residual Ca2+, which temporally and spatially accumulates in the active zone during trains of action potentials, act as potential effectors for these reactions at the presynaptic terminal. Two pieces of evidence are shown in this article (Figures α12.1 subunit at the mouth of Ca2+ channels, senses firing-dependent temporal changes in local and global elevations of Ca2+, and regulates activity of Ca2+ channels for SNAREs-mediated synaptic vesicle exocytosis [2+ channels results in presynaptic depression and facilitation (2+, Ca2+/CaM binds to mGluR4 in place of Munc18-1 [2+/CaM regulation of the mGluR4/Munc18-1 interaction results in presynaptic facilitation. The presynaptic events shown here control synaptic efficacy and encode postsynaptic activity that underlie generation of the synaptic plasticity resulting in regulation of neural circuits.Neuronal firing activity controls the functions of synaptic proteins and dynamically remodels synaptic efficacy. Ca Figures  and 2. Focytosis , 66. Thelitation . Secondllitation . Munc18-Munc18-1 , 62. The