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1.
Reetz  G.  Wiesinger  H.  Reiser  G. 《Neurochemical research》1997,22(5):621-628
Oscillations of cytosolic Ca2+ activity ([Ca2+]i) induced by stimulation with ATP in rat astrocytes in primary cultures were analysed. Astrocytes, prepared from the brains of newborn rats, loaded with the fluorescent Ca2+ indicator fura-2/AM, were continuously stimulated with ATP (10 M). ATP caused a large initial [Ca2+ peak, followed by regular [Ca2+]i oscillations (frequencies 1–5/min). Astrocytes were identified by glial fibrillary acidic protein staining of cells after [Ca2+]i recording. The oscillations were reversibly blocked by the P2 purinoceptor antagonist suramin (30 M). Influx of extracellular Ca2+ and mobilization of Ca2+ from intracellular stores both contributed to the oscillations. The effects of hypertonic and hypotonic superfusion medium on ATP-induced [Ca2+]i oscillations were examined. Hypertonic medium (430 mOsm) reversibly suppressed the ATP-induced oscillations. Hypotonic medium (250 mOsm), in spite of having heterogeneous effects, most frequently induced a rise in [Ca2+]i, or reversibly increased the frequency of the oscillations. Thus, a change in cell volume might be closely connected with [Ca2+]i oscillations in astrocytes indicating that [Ca2+]i oscillations in glial cells play an important role in regulatory volume regulation in the brain.  相似文献   

2.
Extracellular ATP caused a dose-dependent accumulation of inositol phosphates and a rise in cytosolic free Ca2+ ([Ca2+]i) in C6 glioma cells with an EC50 of 60±4 and 10±5 M, respectively. The threshold concentration of ATP (3 M) for increasing [Ca2+]i was approximately 10-fold less than that for stimulating phosphoinositide (PI) turnover. The PI response showed a preference for ATP; ADP was about 3-fold less potent than ATP but had a comparable maximal stimulation (11-fold of the control). AMP and adenosine were without effect at concentrations up to 1 mM. ATP-stimulated PI metabolism was found to be partially dependent on extracellular Ca2+ and Na+ but was resistant to tetrodotoxin, saxitoxin, amiloride, ouabain, and inorganic blockers of Ca2+ channels (Co2+, Mn2+, La3+, or Cd2+). In Ca2+-free medium, ATP caused only a transient increase in [Ca2+]i as opposed to a sustained [Ca2+]i increase in normal medium. The ATP-induced elevation of [Ca2+]i was resistant to Na+ depletion and treatment with saxitoxin, verapamil and nisoldipine, but was attentuated by La3+. The differences in the characteristics of ATP-caused P1 hydrolysis and [Ca2+]i rise suggest that ATP receptors are independently coupled to phospholipase C and receptor-gated Ca2+ channels. Because of the robust effect of ATP in stimulating PI turnover and the apparent absence of P1-purinergic receptors, the C6 glioma cell line provides a useful model for investigating the transmembrane signalling pathway induced by extracellular ATP. The mechanisms underlying the unexpected finding of [Na+]o dependency for ATP-induced PI turnover require further investigation.Abbreviations PI phosphoinositide - [Ca2+]i cytosolic free Ca2+ concentration - PDBu phorbol 12, 13-dibutyrate - PSS physiological saline solution - IP inositol phosphates - IP1 inositol monophosphate - IP2 inositol bisphosphate - IP3 inositol trisphosphate - IP4 inositol (1,3,4,5) tetrakisphosphate - PLC phospholipase C  相似文献   

3.
Although low Na+ is known to increase the intracellular Ca2+ concentration ([Ca2+]i) in cardiac muscle, the exact mechanisms of low Na+-induced increases in [Ca2+]i are not completely defined. To gain information in this regard, we examined the effects of low Na+ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca2+]i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na+-induced increase in [Ca2+]i was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na+ did not alter the ATP-induced increase in [Ca2+]i in the cardiomyocytes. This increase in [Ca2+]i due to low Na+ and elevated KCl was dependent on the extracellular concentration of Ca2+ (0.25–2.0 mM). The L-type Ca2+-channel blockers, verapamil and diltiazem, at low concentrations (1 M) depressed the low Na+, KCl-induced increase in [Ca2+]i without significantly affecting the response to low Na+ alone. The low Na+, high KCl-induced increase in [Ca2+]i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 M), and diltiazem (5 and 10 M) as well as with amiloride (5–20 M), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 M) and thapsigargin (10 and 20 M). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 M). These data suggest that in addition to the sarcolemmal Na+–Ca2+ exchanger, both sarcolemmal Na+–K+ATPase, as well as the sarcoplasmic reticulum Ca2+-pump play prominent roles in the low Na+-induced increase in [Ca2+]i. (Mol Cell Biochem 263: 151–162, 2004)  相似文献   

4.
The ability of the Ca2+-Mg2+ ATPase pump of skeletal SR to produce and maintain a Ca2+ gradient was studied as a function of the ATP/ADP/Pi ratio. The internal free Ca2+ concentration [Ca2+]i was monitored by changes in fluorescence of CTC. Increasing ADP concentrations in the medium reduce the maximal [Ca2+]i concentration achieved. The inclusion or the omission of 4×10–4 M Pi or doubling the absolute ATP and ADP concentrations at a constant ATP/ADP ratio does not affect the level obtained. The level depends primarily on the ATP/ADP ratio. The [Ca2+] concentration shows a 1.5 power dependence on the ATP/ADP ratio. Further, [Ca2+]i achieved at steady state does not depend on whether the pump had been working in the forward or the reverse direction prior to testing. Analysis shows that the levels of Ca2+ achieved are much lower than the levels predicted thermodynamically under the assumption of ideal coupling between Ca2+ transport and ATP hydrolysis with a stoichiometry of 2:1. Under this condition the osmotic energy of the [Ca2+]i/[Ca2+]o ratio was shown to be 48% as large as the free energy of hydrolysis of ATP, giving an overall thermodynamic efficiency of 48%. Analysis shows that maximal steady-state uptake is determined by the balance between the rates of uptake by the pump and rates of leak processes (intrinsic or extrinsic to the pump). Comparison with other studies shows that the [Ca2+]i achieved results in trans-inhibition of the pump by tying up the Ca2+ translocator in the inwardly oriented phosphorylated form. The absence of an effect of Pi can be taken as evidence that the dissociation of Ca2+ from the inwardly oriented translocator on the phosphoylated enzyme must precede the dephosphorylation of the enzyme.  相似文献   

5.
Summary The Ca2– entry pathways in the basolateral plasma membrane of the isolated, nonperfused proximal straight tubule (PST) of rabbit kidney were investigated using fura-2 fluorescence microscopy. Under isotonic conditions, reduction of bath [Ca2–] from 1 mM to 1 M caused intracellular free calcium concentration ([Ca2+]i) to fall close to zero. Treatment with 10 M verapamil, a calcium channel blocker, had a similar effect. Treatment with verapamil or low Ca2+ also induced fluctuations in cell volume. However, isotonic treatment with 10 M nifedipine, a dihydropyridine (DHP)-type calcium channel blocker, did not affect [Ca2+]i or cell volume, indicating that the endogenous Ca2+ entry pathway is verapamil-sensitive but DHP-insensitive. When cells were exposed to hypotonic solutions in the presence of 1 mM Ca2+, they swelled and underwent normal RVD while [Ca2+]i increased transiently to a peak before decreasing to a late phase plateau level above the baseline level (see McCarty, N.A., O'Neil, R.G. 1991.J. Membrane Biol. 123:149–160). When cells were swollen in the presence of verapamil or low bath [Ca2+], RVD was abolished and [Ca2+]i fell well below the baseline during the late phase response. In contrast, when cells were swollen in the presence of nifedipine, RVD and the late phase rise in [Ca2+]i were abolished, but [Ca2+]i did not fall below the baseline level in the late phase, indicating that nifedipine inhibited the swelling-induced Ca2+ entry but that Ca2+ entry by another pathway was undisturbed. It was concluded that PST cells are characterized by two Ca2+ permeability pathways in the basolateral membrane. Under both isotonic and hypotonic conditions, Ca2+ entry occurs at a slow rate via a verapamil-sensitive, DHP-insensitive baseline Ca2+ entry pathway. Cell swelling activates a separate DHP-sensitive, verapamil-sensitive Ca2+ entry pathway, which is responsible for the supply of Ca ions to the Ca2+-dependent mechanism by which cell volume regulation is achieved.  相似文献   

6.
Maitotoxin (MTX) induces an increase of [Ca2+]i and of phosphoinositide breakdown in various cell types. The [Ca2+]i increase followed with fluorescent probes on cell suspensions has been described as slow and lasting, in contrast to the signal induced by calcium ionophores such as ionomycin. MTX effects have been studied on two fibroblastic cell lines, BHK21 C13 and FR 3T3, synchronized by serum deprivation treatment performed in an isoleucine-free medium for BHK21 C13 cells. In BHK21 C13 cells, flow cytometry analysis showed that two stages, G1/S and G2/M, were particularly susceptible to MTX treatment. Scanning laser cytometry demonstrated that calcium response of FR 3T3 fibroblasts followed with Indo-1 varied during the cell division cycle. The [Ca2+]i increase was almost always vertical, but its delay after MTX addition lasted from zero (S and G2/M transition) to 10–20 min (G1) or more (G2). No [Ca2+]i change could be detected during mitosis. The [Ca2+]i response at the S phase was biphasic. These observations suggest that (1) the lasting response described in the literature represents a global cell population effect, and (2) cells are more sensitive to MTX at specific stages of the cell division cycle, which could correspond to periods when calcium signals have been detected in different cell types.Abbreviations MTX maitotoxin - [Ca2+]i intracellular calcium concentration - IP3 inositol triphosphate  相似文献   

7.
Summary This report summarizes our recent work on the role of intracellular Ca2+ ([Ca2+]i) in regulating mammalian ciliary beat frequency (CBF). CBF from a single ovine cilium and [Ca2+]i from the same cell were measured by digital video phase contrast microscopy and fura-2 ratiometric imaging video microscopy, respectively. Cells were stimulated with two exposures to 10 M acetylcholine (ACh). CBF was recorded during the first and [Ca2+]i during the second stimulation. ACh increased [Ca2+]i and CBF transiently with indistinguishable kinetics and, early in culture, even induced [Ca2+]i oscillations and ciliary frequency modulations with the same peak-to-peak time interval. Cells treated with 1 M thapsigargin, an inhibitor of the endoplasmic-reticulum Ca2+-ATPase, showed transient [Ca2+]i and CBF increases, again with similar kinetics, which often remained at an elevated plateau. Application of ACh to cells pretreated with thapsigargin produced decreases in both [Ca2+]i and CBF. Finally, changing extracellular Ca2+-concentrations induced corresponding changes in [Ca2+]i that were associated with kinetically similar CBF changes. These data strongly suggested that [Ca2+]i is a critical signal to regulate CBF in mammalian tracheal epithelial cells. In an initial effort to provide constraints on the number and type of reactions that link changes in [Ca2+]i to changes in CBF, simultaneous recordings of both signals from a single cell were analyzed. Such recordings provided higher resolution of the kinetic responses of CBF and [Ca2+]i to ACh as well as they allowed direct assessment of the coupling between [Ca2+]i and CBF. Simultaneous measurements revealed that [Ca2+]i and CBF were perfectly correlated within the CBF measurement time resolution, except for the period of the fastest changes in both signals during the initial ACh exposure. There, changes in CBF lagged the changes in [Ca2+]i by 1–3 ciliary beat cycles (ca. 150–450 ms).  相似文献   

8.
Adenosine 5′-triphosphate (ATP) is an extracellular signal that regulates various cellular functions. Cellular secretory activities are enhanced by ATP as well as by cholinergic and adrenergic stimuli. The present study aimed to determine which purinoceptors play a role in ATP-induced changes in the intracellular concentration of calcium ions ([Ca2+]i) and in the fine structure of acinar cells of rat lacrimal glands. ATP induced exocytotic structures, vacuolation and an increase in [Ca2+]i in acinar cells. The removal of extracellular Ca2+ or the use of Ca2+ channel blockers partially inhibited the ATP-induced [Ca2+]i increase. U73122 (an antagonist of PLC) and heparin (an antagonist of IP3 receptors) did not completely inhibit the ATP-induced [Ca2+]i increase. P1 purinoceptor agonists did not induce any changes in [Ca2+]i, whereas suramin (an antagonist of P2 receptors) completely inhibited ATP-induced changes in [Ca2+]i. A P2Y receptor agonist, 2-MeSATP, induced a strong increase in [Ca2+]i, although UTP (a P2Y2,4,6 receptor agonist) had no effect, and reactive blue 2 (a P2Y receptor antagonist) resulted in partial inhibition. The potency order of ATP analogs (2-MeSATP > ATP ⋙ UTP) suggested that P2Y1 played a significant role in the cellular response to ATP. BzATP (a P2X7 receptor agonist) induced a small increase in [Ca2+]i, but α,β-meATP (a P2X1,3 receptor agonist) had no effect. RT-PCR indicated that P2X2,3,4,5,6,7 and P2Y1,2,4,12,14 are expressed in acinar cells. In conclusion, the response of acinar cells to ATP is mediated by P2Y (especially P2Y1) as well as by P2X purinoceptors.  相似文献   

9.
Altered cytosolic free calcium concentrations ([Ca2+]i) accompany impaired brain metabolism and may mediate subsequent effects on brain function and cell death. The current experiments examined whether hypoxia-induced elevations in [Ca2+]i are from external or internal sources. In the absence of external calcium, neither KCl depolarization, histotoxic hypoxia (KCN), nor the combination changed [Ca2+]i. However, with external CaCl2 concentrations as small as 13 M, KCl depolarization increased [Ca2+]i instantaneously while hypoxia gradually raised [Ca2+]i. The combination of KCN and KCl was additive. Increasing external calcium concentrations up to 2.6 mM exaggerated the effects of K+ and KCN on [Ca2+]i, but raising medium calcium to 5.2 mM did not further augment the rise. Diminishing the sodium in the media, which alters the activity and perhaps the direction of the Na/Ca exchanger, reduced the increase in [Ca2+]i due to hypoxia, but enhanced the KCl response. The changes in ATP following K+ depolarization, KCN or their combination in the presence of physiological calcium concentrations did not parallel alterations in [Ca2+]i, which suggests that diminished activity of the calcium dependent ATPase does not underlie the elevation in [Ca2+]i. Valinomycin, an ionophore which reduces the mitochondrial membrane potential, elevated [Ca2+]i and the effects were additive with K+ depolariration in a calcium dependent manner that paralleled the effects of hypoxia. Together these results suggest that hypoxia-induced elevations of synaptosomal [Ca2]i are due to an inability of the synaptosome to buffer entering calcium.  相似文献   

10.
Abstract— ATP-induced changes in the intracellular Ca2+concentration ([Ca2+]i) in neuroblastoma glioma hybrid NG108–15 cells were studied. Using the fluorescent Ca2+indicator fura-2, we have shown that the [Ca2+]i increased in response to ATP. ATP at 3 mM caused the greatest increase in [Caz+]i, whereas at higher concentrations of ATP the response became smaller. Two nonhydrolyzable ATP analogues, adenosine 5′-thiotriphosphate and 5′-adenylyl-β, γ-imidodiphosphate, could not trigger significant [Ca2+]i change, but they could block the ATP effect. Other adenine nucleotides, including ADP, AMP, α,β-methylene-ATP, β,γ-methylene-ATP, and 2-methylthio-ATP, as well as UTP and adenosine, all had no effect on [Ca2+]i at 3 mM. In the absence of extracellular Ca2+, the effect of ATP was inhibited totally, but could be restored by the addition of Ca2+ to the cells. Upon removal of Mg2+, the maximum increase in [Ca2+]i induced by ATP was enhanced by about 42%. Ca2+-channel blockers partially inhibited the ATP-induced [Ca2+]i rise. The ATP-induced [Ca2+]i rise was not affected by thapsigargin pretreatment, though such pretreatment blocked bradykinin-induced [Ca2+]i rise completely. No heterologous desensitization of [Ca2+]i rise was observed between ATP and bradykinin. The magnitude of the [Ca2+]i rise induced by ATP increased between 1.5 and 3.1 times when external Na+was replaced with Tris, N-methyl-d -glucamine, choline, or Li+. The addition of EGTA or verapamil to cells after their maximum response to ATP immediately lowered the [Ca2+]i to the basal level in Na+-containing or Na+-free Tris solution. Our results suggest that ATP stimulates Ca2+influx via at least two pathways: ion channels that are permeable to Ca2+ and Na+, and pores formed by ATP4-.  相似文献   

11.
Y. Iwadate  M. Kikuyama  H. Asai 《Protoplasma》1999,206(1-3):11-19
Summary Trichocyst discharge, ciliary reversal, and cell body contraction inParamecium spp. have all been claimed to be regulated by the intracellular Ca2+ concentration ([Ca2+]i) at the cortical region of the cell. We injected caged Ca2+ intoP. caudatum cells and applied ultraviolet (UV) light to the cell for 125 ms. This did not induce trichocyst discharge but did induce both ciliary reversal and cell body contraction. A re-application of UV for 125 ms triggered trichocyst discharge. These results demonstrate that (1) trichocyst discharge and ciliary reversal and cell body contraction are controlled by [Ca2+]i and (2) the threshold of [Ca2+]i for trichocyst discharge is higher than those for the other two functions.Abbreviations DTT dithiothreitol - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ICL infraciliary lattice - [Ca2+]i intracellular Ca2+ concentration - NP-EG o-nitrophenyl EGTA - PMT photomultiplier tube - UV ultraviolet  相似文献   

12.
The accumulation of the -amyloid peptide (AP) in the brain, produced from the ubiquitously expressed amyloid precursor protein (APP) is a defining feature of Alzheimer's disease (AD). Consistent with studies demonstrating the importance of skin biopsy in the diagnosis of neurodegenerative disorders, we investigated whether differences in intracellular free calcium levels ([Ca2+]i) of cultured cutaneous fibroblasts derived from sporadic AD patients and from age-matched control individuals might be present. [Ca2+]i was measured in Fura-2AM-loaded human fibroblasts by dual wavelength spectrofluorimetry. AD cells exhibited lower [Ca2+]i as compared to the control cultures. Exposure of fibroblasts to AP resulted in increased [Ca2+]i of the control cells, but not of AD fibroblasts. Our test could prove useful in supporting the diagnosis of (sporadic) AD in patients suspected of suffering from the disease.  相似文献   

13.
The thermodynamic efficiency of the calmodulin-activated form of the Ca2+-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca2+ concentration ([Ca2+]i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][Pi]) ratio for the following experimental conditions: 250mM sucrose, 100mM KCI, 0.1mM Mg2+, 25mM HEPES, 25mM Tris, pH 7.40, at 37°C, [Ca2+]o=50nM (1mM Ca/EGTA buffer), 0.75mM Mg-ATP, 0.1mM Pi, variable [ADP]. Under these conditions, with the pump working near itsK m of 64nM, the [Ca2+]i achieved was 18mM, decreasing with increasing [ADP] for [ADP] 0.84mM. A plot of the square of the [Ca2+]i/[Ca2+]o ratio against [ATP]/([ADP][Pi]) gave a straight line with a slope of 1.5×107M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09×107M). These results demonstrate (1) tight coupling between Ca2+ transport and ATP hydrolysis with a stoichiometry of 2 Ca2+ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (<0.83mM) showed the unexpected result that ADP increases the rate of theforward reaction of the pump. The maximal effect on the initial rate is a 96±5% increase, with an EC50 of approximately 0.4mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed.  相似文献   

14.
Using the patch clamp technique, we have characterized a small conductance, calcium-activated potassium (SK) channel in the C6 glioma cell line. Elevation of cytosolic Ca2+ concentration ([Ca2+] i ) by applications of serotonin or ionomycin induced bursts of channel openings recorded in the cell-attached configuration. These channels underlie the serotonin-induced, [Ca2+] i -activated whole-cell K+ conductance described previously. [Ca2+] i directly activated SK channels in inside-out patches with a biphasic concentration dependence. Submicromolar [Ca2+] i induced bursts of channel openings with a unitary conductance of about 25 pS, similar to that of the serotonin-induced channels. Supramicromolar [Ca2+] i caused prolonged openings with a unitary conductance of about 35 pS, resulting in a pronounced increase of the average current in patches exposed to [Ca2+] i above 100 m. The two modes of opening reflect the activity of the same SK channel. The channel conductance depended on external K+ concentration with K Dof 5 m. The channel was slightly permeable to cations other than K+, with a permeability ratio for K+Ca2+Na+ of 10.0400.030, respectively. ATP was required to maintain channel activity in outside-out patches but was not essential in inside-out patches. The modulation of SK channels in C6 cells by components in their microenvironment may be related to the role of glial cells in controlling the extracellular milieu in the CNS.The authors are grateful to Dr. M. Segal for continuous support, stimulating discussions and criticism throughout the course of this work, to Dr. I. Steinberg for helpful suggestions and to Dr. H. Jarosch, for helping with the Fortran application. N.M.'s research was supported in part by BARD, the U.S.-Israel Binational Agricultural Research and Development Fund, grant no. IS-1670-89RC.  相似文献   

15.
Alterations in intracellular free calciumconcentration ([Ca2+]i) areinstrumental in apoptosis. We have previously shown that a[Ca2+]i increase above 1000 nM isrelated to the appearance of apoptosis in serum-free cultures ofgranulosa cell sheets. In the present study we examined how the[Ca2+]i increase relates toindicators of distinct phases of the apoptotic cascade. We used adouble staining technique whereby loading with theCa2+ indicator fura-2 and capture of a[Ca2+]i image, was followed bystaining with annexin-V, as an early apoptotic marker or withacridine orange, marking the late degradation phase. Calcium imagingshowed a large heterogeneity of cellular[Ca2+]i levels. [Ca2+]i was moderately increased to230 nM in annexin positive cells but was at resting levelin cells with nuclear manifestations of apoptosis as evidenced byacridine orange. Our results suggest that a moderate[Ca2+]i increase is related tophosphatidylserine translocation and that[Ca2+]i has already recovered inapoptotic cells displaying chromatin condensation and/or nuclearfragmentation. Granulosa cells with[Ca2+]i above 1000 nM were neverobserved to stain positive for the apoptotic markers used; therefore,large [Ca2+]i increases areprobably related to the apoptosis initiation phase occurring upstreamof phosphatidylserine exposure.  相似文献   

16.
Intracellular Ca2+ mobilization events were assessed in mouse L cells, which contain native prostaglandin E1 receptors and transfected human 2 adrenergic receptors. Both Fura2 (single cell measurements) and Quin 2, (cuvette assays) were used to determine [Ca2+]i levels. Our results demonstrate that in the transfected cells there is a dose-dependent increase in [Ca2+]i in response to isoproterenol (0.1 nM–100 nM), which is inhibited by the -adrenergic antagonist, propranolol, and is a result of intracellular Ca2+ release. [Ca2+]1 in these cells was also increased by prostaglandin E1, 8 bromo cyclic AMP, and aluminum fluoride. Both 8 bromo cAMP and isoproterenol induced a rapid increase in the levels of IP1, IP2, and IP3. The data presented demonstrate that the elevation of intracellular cyclic AMP induces an increase in IP3 production which leads to an elevation in [Ca2+];. We propose that this cyclic AMP dependent activation of the IP3 generating system occurs at a post-receptor site.Abbreviations cAMP Adenosine Cyclic 3-5-Monophosphate - [Ca2+]i intracellular [Ca2+]i - 8 Br cAMP 8 Bromo Adenosine Cyclic 3-5-Monophosphate - DAG Diacylglycerol - EGTA] [Ethylene Bis (oxyethylenenitrilo)] Tetracetic acid - BSA Bovine Serum Albumin - HBSS-H Hanks' Balanced Salt Solution buffered with HEPES to pH 7.4 - HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - PIP2 Phosphatidylinositol 4,5-bisphosphate - IP2 Inositol 4 Phosphate - IP2 Inositol 4,5 Bisphosphate - IP3 Inositol Trisphosphate - PGE1 Prostaglandin E1 - PBS Phosphate Buffered Saline Solution  相似文献   

17.
Fedirko  N. V.  Klevets  M. Yu.  Kruglikov  I. A.  Voitenko  N. V. 《Neurophysiology》2001,33(4):216-223
Using a Ca2+-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K+] e up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca2+] i , whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA2+-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca2+] i and a decrease in the K+-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca2+] i . Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca2+ influx and Ca2+-induced Ca2+ release from the endoplasmic reticulum; the initial level of [Ca2+] i was restored at the joint involvement of Ca2+-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria.  相似文献   

18.
We investigated whether amyloid--peptide (A1–42) has an effect on the elevations of the intracellular concentration of Ca2+ ions ([Ca2+]i) induced by depolarizations of NG108-15 cells and on related Ca2+ channels. A1–42 (10-1000 nM) had no immediate effect on depolarization-induced [Ca2+]i elevations. [Ca2+]i increases were slightly diminished in cells grown in the presence of 100 or 1000 nM A1–42. Nifedipine (1 M) reduced these elevations equally in cells grown in the absence or presence of A1–42. In contrast, the ability of -conotoxin GVIA to diminish the depolarization-induced [Ca2+]i responses became lost in cells grown in the presence of 100 nM A1–42. This indicates that the influx of calcium through the N-type Ca2+ channels was compromised by the chronic exposure of cells to a submicromolar concentration of A1–42, presumably because of impairement of their function or diminished expression. This may be important in the pathogeny of Alzheimer's dementia in view of the pivotal role of N-type Ca2+ channels in neurotransmitter release.  相似文献   

19.
The effects of extracellular Mg2+ on both dynamic changes of [Ca2+]i and apoptosis rate were analysed. The consequences of spatial and temporal dynamic changes of intracellular Ca2+ on apoptosis, in thapsigargin- and the calcium-ionophore 4BrA23187-treated MCF7 cells were first determined. Both 4BrA23187 and thapsigargin induced an instant increase of intracellular Ca2+ concentrations ([Ca2+]i) which remained quite elevated (> 150 nM) and lasted for several hours. [Ca2+]i increases were equivalent in the cytosol and the nucleus. The treatments that induced apoptosis in MCF7 cells were systematically associated with high and sustained [Ca2+]i (150 nM) for several hours. The initial [Ca2+]i increase was not determinant in the events triggering apoptosis. Thapsigargin-mediated apoptosis and [Ca2+]i rise were abrogated when cells were pretreated with the calcium chelator BAPTA. The role of the extracellular Mg2+ concentration has been studied in thapsigargin treated cells. High (10 mM) extracellular Mg2+, caused an increase in basal [Mg2+]i from 0.8 ± 0.3 to 1.6 ± 0.5 mM. As compared to 1.4 mM extracellular Mg2+, 1 M thapsigargin induces, in 10 mM Mg2+, a reduced percentage from 22 to 11% of fragmented nuclei, a lower sustained [Ca2+]i and a lower Ca2+ influx through the plasma membrane. In conclusion, the cell death induced by thapsigargin was dependent on high and sustained [Ca2+]i which was inhibited by high extracellular and intracellular Mg2+.  相似文献   

20.
1. The elevation of intracellular Ca2+ levels ([Ca2+]i) in immortalized hypothalamic neurons (GT1–7 cells) after exposure to Alzheimer's ß-amyloid protein (AßP[25–35]) was investigated using a multisite fluorometry system.2. The marked rise in [Ca2+]i appeared afterexposure to 5–20-M AßP[25–35]. Analysis of the spatiotemporal patterns of [Ca2+]i changes revealed that the magnitude and the latency of the response to AßP in each cell werehighly heterogeneous.3. The preadministration of 17ß-estradiol, 17-estradiol, phloretin and cholesterol, which influence the properties of membranes, such as membrane fluidity or membrane potential, significantly decreased the rise in [Ca2+]i.4. These findings support the idea that disruption of calcium homeostasis by AßP channels may be the molecular basis of the neurotoxicity of AßP and of the pathogenesis of Alzheimer's disease. It is also suggested that membrane properties may play key roles in the expression of neurotoxicity.  相似文献   

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