However, low dose extracellular ATP induces Ca 2+ shifts if cells express members of P2X and P2Y trans- porter family, as it is the case in HeLa cells . Therefore, in this specific cell line and setting, we cannot rule out the occurrence of false-positives. Falsely categorizing a substance as positive or negative due to specific characteristics of the tested cells is al- ways a risk in cytotoxicity screens. For example bleo- mycin, a well-established clastogenic agent and anti- tumor drug has to be taken up via the hCT2-transporter, which is the rate-limiting step determining its toxic ac- tivity as reviewed recently . To avoid false-negative and false-positive results we suggest testing a panel of cell lines, which differ in their receptor repertoire. It can be expected that physiological molecules will obviously induce cellular responses including Ca 2+ dependent signaling processes. In contrast, engineered substances inducing a rise in free cytosolic Ca 2+ as pre- sented in this study are indicative of unwanted bio- logical effects. Therefore we conclude that cytosolic Ca 2+ increases within the first 5 s of exposure as mea- sured with Fluo-4 dye are predictive of the cytotoxic po- tential of a xenobiotic compound.
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To experimentally evaluate whether store operated cal- cium entry (SOCE) plays a role in calcium homeostasis in the HEK293 cell response to Echinacea-extracts, external Ca 2+ was removed and internal Ca 2+ stores were depleted using the sarcoplasmic/ER Ca 2+ -ATPase (SERCA) pump inhibitor, thapsigargin. Restoration of external Ca 2+ following thapsigargin-treatment indicates the function of SOCE, which can be observed as an increase in cytosolic Ca 2+ . Here, the effect of thap- sigargin is compared to the effect of Echinacea extract. The Ca 2+ responses evoked by Echinacea and thapsigar- gin are similar (Figure 6). In the absence of extracellular Ca 2+ , both agents evoke a transient intracellular Ca 2+ elevation, interpreted as being due to Ca 2+ release from internal stores. After Ca 2+ -containing medium is intro- duced, a sustained increase in cytosolic Ca 2+ is observed for both treatments (Figure 6A, 6B). This pattern is
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disease. The cytosolic Ca2+ concentration ([Ca2+]i) at rest and the increase of [Ca2+]i after addition of acetylcholine are the same in cultured muscle cells of controls and patients. The half-life of the maximal response, however, is raised three times in the pathological muscle cells. Addition of dantrolene or verapamil after the maximal response accelerates the
electrolyte transport was associated with an abrupt increase in the permeability of the monolayer. Cyclic AMP and cyclic GMP in the T84 monolayers were not increased by the bile salt, but in the presence of extracellular Ca2+, free cytosolic Ca2+ increased with a graded dose effect and time course that corresponded approximately to the changes in short circuit current (Isc). The results suggest that luminal bile salts at a relatively high
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homeostasis and function. To examine the influence of cytosolic Na+ on Ca2+ regulation in human peripheral blood lymphocytes, Ca2+ entry and cytosolic Ca2+ (measured with fura- 2) were monitored in cells in which cytosolic Na+ was increased and/or the Na+ gradient was decreased by reduction of external Na+ concentration. Ouabain-treated cells (0.1 mM for 30 min at 37 degrees C), suspended in Na(+)-free medium, showed a 30-65% increase in Ca2+ uptake compared to cells in 140 mM Na+ medium. Enhanced Ca2+ influx was entirely dependent on ouabain pretreatment and reversal of the Na+ gradient. Na pump inhibition or Na ionophore addition and subsequent exposure to Na(+)-free medium resulted in a sustained elevation of cytosolic Ca2+. As preincubation of cells in Ca(2+)-free medium further enhanced the ouabain-dependent increase in cytosolic Ca2+, the effects of the microsomal Ca(2+)-ATPase inhibitor thapsigargin on Ca2+ influx and cytosolic Ca2+ were studied. Thapsigargin stimulated Ca2+ entry following ouabain pretreatment and reversal of the Na+ gradient; the effects of thapsigargin were retained in the presence of LaCl3, a
Using a glucose-responsive beta cell line, we tested the hypothesis that the free cytosolic Ca2+ concentration ([Ca2+]i) is the primary signal that couples a stimulus to insulin secretion, and examined the involvement of the extracellular Ca2+ pool in this process. Glucose or depolarization of the beta cell with 40 mM K+ stimulated a monophasic release of insulin directly proportional to the extracellular Ca2+ concentration. 40 mM K+ increased 45Ca2+ uptake and increased [Ca2+]i, which was measured with quin 2, 4.7-fold, from 56 +/- 3 to 238 +/- 17 nM. With high glucose, 45Ca2+ uptake did not increase, and [Ca2+]i was unchanged or fell slightly. There was a striking correlation between inhibitory effects of verapamil, the Ca2+ channel blocker, on insulin secretion and the rise in [Ca2+]i evoked by K+. Higher concentrations of verapamil were required to inhibit glucose- than K+-stimulated insulin secretion (dose giving half-maximal effect of 1.4 X 10(-4) M vs. 6.0 X 10(-7) M). Incubation in Ca2+-free, 1 mM EGTA buffer for 30 min lowered [Ca2+]i to 14 +/- 2 nM, and inhibited acute insulin release to both secretagogues. If high glucose was present in the Ca2+-free period, reintroduction of 2.5 mM Ca2+ in high glucose restored insulin secretion only to the basal rate. However, if low glucose was present during the Ca2+-free period, high glucose and 2.5 […]
from intracellular stores (Berridge et al., 2000). As electrically inexcitable cells, glial cells use this pathway in response to many kinds of stimuli, including neurotransmitters, neuromodulators, growth factors and cytokines (Lohr and Deitmer, 2006; Fiacco and McCarthy, 2006). In the nervous system, glial cells often respond to active neurons releasing neurotransmitters, which activate metabotropic receptors in the glial cells. Recent evidence suggests that glial cells may themselves release transmitters such as glutamate and ATP, mediated by cytosolic Ca 2+ transients (Araque et al., 2001; Pascual et al., 2005). Due to the presence of several G proteins, which initiate or suppress different signalling cascades (for a review, see Luttrell, 2006), we tried to elucidate the dominant pathways initiated by global activation of G protein.
and the cis (cytosolic) side contained 100 mM Tris/HEPES (pH 7.35). The cis chamber was held at virtual ground and the trans chamber was voltage clamped (BC-525C bilayer clamp; Warner Instruments) to 0 mV, +10 mV, and –10 mV as indicated (Figure 1C). The current across the BLM was amplified (OC-725C), filtered at 5 kHz, digitized (Digidata 1200; Axon Instruments), and stored on a computer hard drive and recordable opti- cal discs. For presentation, the current traces were digitally filtered at 200 Hz (pClamp 6.0; Axon Instruments). For off-line computer analysis, stationary noise analysis method was used as previously described (10). Using WinEDR version 2.4.3 software (BioLogic) (46), the currents were filtered at 100 Hz and the mean current (I) and the current variance (δ 2 )
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indicator of fertilization was the secretion of cell wall components, imaged using 2-photon microscopy of the cellulose stain CFW. CFW staining was evident within 15 minutes of sperm and egg mixing, and propagated across the egg within 5 minutes. (B) Sperm pronuclear (‘sp’) motion towards the egg pronucleus (‘ep’), visualized using 2-photon microscopy of Hoechst 33342-labelled sperm. This representative series of images shows the sperm pronucleus as a light blue dot against a green autofluorescent background. The final image was just before pronuclear fusion. (C) Time courses during the first 3 hours after fertilization for cell wall secretion (upper plot, grey circles, n=8), sperm pronuclear migration (upper plot, white circles, n=10), cytosolic [Ca 2+ ]
The signaling events that trigger parasite egress remain poorly defined. Ca 2⫹ ionophores have long been known to stimulate egress in T. gondii (64). However, more recently, phosphodiesterase inhibitors were shown to have similar effects (20, 61). Our experiments revealed a strong and rapid FIGURE 6. Enh1 elicits asynchronous cytosolic Ca 2ⴙ fluxes and blocks zaprinast-induced egress. A, video microscopy of GCaMP6f-expressing parasites treated with Enh1 or zaprinast. Time after the addition of the compound is indicated. Different times were used to capture the fast and slow responses of zaprinast and Enh1, respectively. B, kymographs illustrate average fluorescence intensities of individual parasites, per row, during the course of the treatment indicated. Black indicates that parasites egressed from vacuoles. C, change in fluorescence of the parasites illustrated in B over the 40 s following the addition of zaprinast. Measurements from each biological replicate are colored separately. Mean change for each group is indicated with a horizontal line. ****, p ⬍ 0.0001, two-tailed t test.
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The mechanism by which cholecystokinin (CCK) regulates human pancreatic function has not yet been clearly clarified, partly because of obvious difficulties in sampling normal pancreatic tissue from humans (Miller, 1996). Extensive studies of mouse and rat pancreatic acinar cells have shown that physiological concentrations (1-20 pM) of CCK, like acetylcholine (ACh), elicit cytosolic Ca 2+ signals that control secretion (Petersen et al, 2008). Such signals result from a direct action of CCK on CCK receptors in the pancreatic acinar cell plasma membrane (Jensen et al, 1989), and in vivo, from vagal (neuronal) afferents stimulating acinar cell muscarinic receptors through vagal loops (Li et al, 1993). Failure to show functional responses to CCK in human pancreatic acinar cells (Ji et al, 2001), however, has led to the conclusion that CCK only stimulates human pancreatic acinar cells through vagal afferents (Ji et al, 2001, Owang et al, 2004, Dufresne et al, 2006).
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Bone is constantly being remodeled by the delicate bal- ance between the activities of osteoblasts, which are in charge of bone mineralization, and osteoclasts, which re- sorb bone matrix. In the process of bone remodeling, re- ceptor activator of nuclear factor-κB ligand (RANKL), a key molecule expressed in osteoblasts, mediates osteoclas- togenesis resulting in breakdown of bone. Contact be- tween RANKL and receptor activator of nuclear factor-κB (RANK), expressed on osteoclast precursor cells, mediates differentiation-related signals through nuclear factor of ac- tivated T-cell c1 (NFATc1) activation . Notably, re- peated reports have clearly showed that RANKL-induced free cytosolic Ca 2+ ([Ca 2+ ] i ) oscillations modulate NFATc1
To study the mediation of Ca2+ influx by second messengers in myeloid cells, we have combined the whole-cell patch clamp technique with microfluorimetric measurements of [Ca2+]i. Me2SO-differentiated HL-60 cells were loaded with the fluorescent Ca2+ indicator Indo-1, allowed to adhere to glass slides, and patch-clamped. Receptor agonists and Ca(2+)-ATPase inhibitors were applied by superfusion and inositol phosphates by
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An inverted microscope (TMD, Nikon, Tokyo) equipped with a laser confocal scanner unit (Insight Plus-IQ, Meridian Instruments Far East, Inc., Tokyo) was used with excitation at a wavelength of 488 nm (Fig. 1). The magnified image of the specimen was recorded with a video recorder (BR-S811, Victor, Tokyo) through a CCD camera (HCC-600M, Flovel, Tokyo). The recorded images were transferred to a computer (Apple Macintosh 7100) and analyzed. Relative cytosolic [Ca 2+ ] was expressed as F/F
molecules are sufficiently large to establish the deter- ministic concentration profile on the time scale of typ- ical channel state changes due to the frequent sampling of space by thermal motion. The number of SERCA molecules is orders of magnitude larger than the num- ber of Ca 2+ channels. Hence, we can describe diffusion, the reactions involving cytosolic Ca 2+ binding molecules and the SERCA flux by reaction-diffusion equations like Eq. (1). The opening and closing of channels causes time dependent source terms in the partial differential equa- tion for the Ca 2+ concentration. We illustrate that with a simple model comprising cytosolic Ca 2+ c, one Ca 2+ buffer b (Ca 2+ bound form) and the ER Ca 2+ concentra-
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controlling event. However, although the concept of Ca 2+ control of exocytosis dates back over 20 years (Douglas, 1968), only recently developed methodological approaches have provided a proper quantitative evaluation of the role Ca 2+ plays in this process. For example, in adrenal chromaffin cells, the use of video-imaging techniques to visualise the stimulus-induced changes in the concentration of cytosolic free Ca 2+ ([Ca 2+ ] i ) in Fura-2-
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understood. The evidence in squid axons suggests a phosphorylation mechanism (DiPolo and Beaugé, 1993), but antiport current measurements in cardiac sarcolemmal patches (Collins et al. 1992; Hilgemann and Collins, 1992) do not support this hypothesis. The latter studies suggest that the ATP effect is indirect and is mediated by an aminophospholipid translocase that maintains an elevated concentration of acidic phospholipids at the cytosolic surface of the membrane bilayer. This mechanism is consistent with the properties of the isolated antiporter, since activity is known to be stimulated by negatively charged amphiphiles, such as phosphatidylserine (Collins and Hilgemann, 1993).
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calcium ([Ca2+]c) decline (tau Ca) was increased during low flow ischemia, and if there was a relationship between the time constant of left ventricular pressure decline (tau P) and tau Ca. Isolated perfused hearts were studied using indo-1 fluorescence ratio as an index of [Ca2+]c.tau P was used as an index of myocardial relaxation. The time constant of decline of the indo-1 ratio increased from 74 +/- 5 ms to 95 +/- 4, 144 +/- 10, and to 204 +/- 16 ms when coronary flow was reduced was reduced to 50, 20, and 10% of control, respectively. Indo-1 transients were calibrated to calculate tau Ca. tau Ca increased from 67 +/- 6 ms to 108 +/- 9 and 158 +/- 19 ms when coronary flow was reduced to 20 and 10% of control, respectively. There was a linear relationship between tau Ca and tau P (r = 0.82). These data support the hypothesis that during low flow ischemia, impaired myocardial relaxation may be caused by slowing of [Ca2+]c decline.
accompanied by a fall in pHi (delta pHi, -0.064 +/- 0.0085 P < 0.01, and -0.05 +/- 0.012 pH units, P < 0.01, respectively). Neither the fall in pHi nor the rise in Cai2+ elicited by Ang II was prevented by pretreatment with agents which block the action of this agonist on pHi via the stimulation of the Cl/HCo3 exchangers (DIDS, 50 microM) or the Na+/H+ antiporter (EIPA, 50 microM). In the presence of DIDS and EIPA, Ang II produced a fall in pHi (delta pHi, -0.050 +/- 0.014, P < 0.01) and a rise in Cai2+ (delta Ca2+ 252 +/- 157 nM, P < 0.01). That the change in pHi was secondary to changes in Cai2+ was inferred from the finding that, when the rise in Cai2+ elicited by Ang II was prevented by preincubation with […]
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Microsomal Ca(2+)-ATPase inhibitors such as thapsigargin (THG), cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-hydroquinone (DBHQ) have been shown to inhibit Ca2+ reuptake by the intracellular stores and increase cytosolic free Ca2+ ([Ca2+]i). DBHQ is a commercially available non-toxic synthetic compound chemically unrelated to THG and CPA. In this study, we tested the feasibility of utilizing DBHQ to improve Cl- secretion via the Ca(2+)-dependent pathway, in the cystic fibrosis (CF)-derived pancreatic epithelial cell line CFPAC-1. DBHQ stimulated 125I efflux and mobilized intracellular free Ca2+ in a dose- dependent manner. The maximal effects were seen at concentrations of 25-50 microM. DBHQ (25 microM) caused a short-term rise in [Ca2+]i in the absence of ambient Ca2+, and a sustained elevation of [Ca2+]i in cell monolayers bathed in the efflux solution (1.2 mM Ca2+), which was largely attenuated by Ni2+ (5 mM). Bath-application of DBHQ induced an outwardly-rectifying whole-cell Cl- current, which was abolished by pipette addition of BAPTA (5 mM) or CaMK [273-302] (20 microM), an inhibitory peptide of multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII). Pretreatment of monolayers of CFPAC-1 cells with DBHQ for 4-5 min significantly increased the Ca(2+)-independent or autonomous activity of CaMKII assayed in the cell homogenates. Thus, DBHQ appears to enhance Cl- channel activity via a Ca(2+)-dependent mechanism involving CaMKII. Pretreatment of CFPAC-1 cells with up to 50 microM DBHQ for 6 h […]