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1.
The mechanism underlying the generation of cytosolic free Ca2+ ([Ca2+i) oscillations by bombesin, a receptor agonist activating phospholipase C, in insulin secreting HIT-T15 cells was investigated. At 25 μM, 61% of cells displayed [Ca2+]i oscillations with variable patterns. The bombesin-induced [Ca2+]i oscillations could last more than 1 h and glucose was required for maintaining these [Ca2+ fluctuations. Bombesin-evoked [Ca2+]i oscillations were dependent on extracellular Ca2+ entry and were attenuated by membrane hype rpolarization or by L-type Ca2+ channel blockers. These [Ca2+]i oscillations were apparently not associated with fluctuations in plasma membrane Ca2+ permeability as monitored by the Mn2+ quenching technique. 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) and 4-chloro-m-cresol, which interfere with intracellular Ca2+ stores, respectively, by inhibiting Ca2+-ATPase of endoplasmic reticulum and by affecting Ca2+-induced Ca2+ release, disrupted bombesin-induced [Ca2+]i oscillations. 4-chloro-m-resol raised [Ca2+]i by mobilizing an intracellular Ca2+ pool, an effect not altered by ryanodine. Caffeine exerted complex actions on [Ca2+]i It raised [Ca2+]i by promoting Ca2+ entry while inhibiting bombesin-elicited [Ca2+]i oscillations. Our results suggest that in bombesin-elicited [Ca2+]i oscillations in HIT-T15 cells: (i) the oscillations originate primarily from intracellular Ca2+ stores; and (ii) the Ca2+ influx required for maintaining the oscillations is in part membrane potential-sensitive and not coordinated with [Ca2+]i oscillations. The interplay between intracellular Ca2+ stores and voltage-sensitive and voltage-insensitive extracellular Ca2+ entry determines the [Ca2+]i oscillations evoked by bombesin.  相似文献   

2.
DMSO differentiated U937 cells responded to 10−6 M LTD4, LTB4 and FMLP with an increase in both InsP formation and [Ca2+]i. FMLP caused a greater rise in InsPs than either LTD4 or LTB4, which were equivalent. LTD4, however, caused a greater increase in [Ca2+]i than LTB4 (4-fold) or FMLP. The FMLP [Ca2+]i and InsP responses were abolished by pertussis toxin (100 ng/ml for 4 h) but were unaffected by PMA (10−7 M for 3 min). In contrast, the LTD4 [Ca2+]i and InsP responses were reduced by only 50% by pertussis toxin, whilst PMA reduced the [Ca2+]i and InsP responses to LTD4 by 75 and 30%, respectively. These results suggest that mechanisms additional to InsP formation exist for mediating LTD4 evoked increases in [Ca2+]i.  相似文献   

3.
The alkaloid derivative vinpocetine (14-ethoxycarbonyl-(3,16-ethyl)-14,15-eburnamine; Cavinton) has a well known beneficial effect on brain function in hypoxic and ischemic conditions. While it increases CNS blood flow and improves cellular metabolism, relatively little is known about vinpocetine's underlying molecular mechanisms on the single cell level. Since apoptotic and necrotic cell damage is always preceded by an increase in [Ca2+]i, this study investigated the effect of vinpocetine on [Ca2+]i increases in acute brain slices. Sodium influx is an early event in the biochemical cascade that takes place during ischemia. The alkaloid veratridine can activate this Na+ influx, causing depolarization and increasing [Ca2+]i in the cells. Therefore, it can be used to simulate an ischemic attack in brain cells. Using a cooled CCD camera-based ratio imaging system and cell loading with fura 2/AM, the effect of vinpocetine on [Ca2+]i changes in single pyramidal neurons in the vulnerable CA1 region of rat hippocampal slices was investigated. Preperfusion and continuous administration of vinpocetine (10 M) significantly inhibited the elevation in [Ca2+]i induced by veratridine (10 M). When the drug was administered after veratridine, it could accelerate the recovery of cellular calcium levels. Piracetam, another nootropic used in clinical practice, could attenuate the elevation of [Ca2+]i only at a high, 1 mM, concentration. We have concluded that vinpocetine, at a pharmacologically relevant concentration, can decrease pathologically high [Ca2+]i levels in individual rat hippocampal CA1 pyramidal neurons; this effect might contribute to the neuroprotective property of the drug.  相似文献   

4.
Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca2+]i) in a dose-dependent manner and activated the L-type Ca2+ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca2+]i elevation was abolished in the presence of the ETA receptor blocker BQ123, but was not affected by the ETB receptor blocker BQ788. ET-1-induced an increase in [Ca2+]i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca2+]i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca2+ channel current and an increase of open-state probability (NPo) of an L-type single Ca2+ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca2+ channel activation and Ca2+-induced Ca2+ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).  相似文献   

5.
Summary Calcium signaling systems in nonexcitable cells involve activation of Ca2+ entry across the plasma membrane and release from intracellular stores as well as activation of Ca2+ pumps and inhibition of passive Ca2+ pathways to ensure exact regulation of free cytosolic Ca2+ concentration ([Ca2+] i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca2+ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca2+ entry and release while bradykinin appeared only to release Ca2+ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca2+] i response to these agonists was examined by four methods. Low concentrations of TPA (2×10–10 m) had no effect on Ca2+ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca2+] i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca2+] i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca2+ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca2+ entry, fura-2 loaded cells were suspended in Mn2+ rather than Ca2+ buffers. Addition of EGF or TGF- resulted in Ca2+ release and Mn2+ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn2+ influx. Thus, PKC is able to regulate Ca2+ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10–8 m) inhibited not only Ca2+ entry but also Ca2+ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca2+ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca2+] i , showing a common pool of Ca2+ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca2+] i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca2+ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca2+] i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca2+ for EGF-mediated Ca2+ release which also respond differently to TPA.  相似文献   

6.

Background

Extracellular matrix (ECM) components and intracellular pH (pHi) may serve as regulators of cell migration in various cell types.

Methods

The Oris migration assay was used to assess the effect of fibronectin (FN) on cell motility. The Na+/H+ exchanger (NHE)-1 activity was evaluated by measuring pHi and [22Na+] uptake. To examine activated signaling molecules, western blot analysis and immunoprecipitation was performed.

Results

ECM components (FN, laminin, fibrinogen, and collagen type I) increased [22Na+] uptake, pHi, and cell migration. In addition, FN-induced increase of cell migration was inhibited by NHE-1 inhibitor amiloride or NHE-1-specific siRNA. FN selectively increased the mRNA and protein expression of NHE-1, but not that of NHE-2 or NHE-3. FN binds integrin β1 and subsequently stimulates caveolin-1 phosphorylation and Ca2 + influx. Then, NHE-1 is phosphorylated by RhoA and Rho kinases, and Ca2 +/calmodulin (CaM) signaling elicits complex formation with NHE-1, which is enriched in lipid raft/caveolae microdomains of the plasma membrane. Activation of NHE-1 continuously induces an increase of [22Na+] uptake and pHi. Finally, NHE-1-dependent extracellular signal-regulated kinase (ERK) 1/2 phosphorylation enhanced matrix metalloproteinase-2 (MMP-2) and filamentous-actin (F-actin) expression, partially contributing to the regulation of embryonic stem cells (ESCs) migration.

Conclusions

FN stimulated mESCs migration and proliferation through NHE-1 activation, which were mediated by lipid raft-associated caveolin-1, RhoA/ROCK, and Ca2 +/CaM signaling pathways.

General significance

The precise role of NHE in the modulation of ECM-related physiological functions such as proliferation and migration remains poorly understood. Thus, this study analyzed the relationship between FN and NHE in regulating the migration of mouse ESCs and their related signaling pathways.  相似文献   

7.
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   

8.
9.

Background

All identified mammalian TRPC channels show a C-terminal calmodulin (CaM)- and inositol 1,4,5-trisphosphate receptors (IP3Rs)-binding (CIRB) site involved in the regulation of TRPC channel function.

Objectives

To assess the basis of CaM/IP3Rs binding to the CIRB site of TRPC6 and its role in platelet physiology.

Methods

Protein association was detected by co-immunoprecipitation and Western blotting, Ca2+ mobilization was measured by fluorimetric techniques and platelet function was analyzed by aggregometry.

Results

Co-immunoprecipitation of TRPC6 with CaM or the IP3Rs at different cytosolic free Ca2+ concentrations ([Ca2+]c) indicates that the association between these proteins is finely regulated by cytosolic Ca2+ via association of CaM and displacement of the IP3Rs at high [Ca2+]c. Thrombin-stimulated association of TRPC6 with CaM or the IP3Rs was sensitive to 2-APB and partially inhibited by dimethyl BAPTA loading, thus suggesting that the association between these proteins occurs through both Ca2+-dependent and -independent mechanisms. Incorporation of an anti-TRPC6 C-terminal antibody, whose epitope overlaps the CIRB region, impaired the dynamics of the association of TRPC6 with CaM and the IP3Rs, which lead to both inhibition and enhancement of thrombin- and thapsigargin-evoked Ca2+ entry in the presence of low or high, respectively, extracellular Ca2+ concentrations, as well as altered thrombin-evoked platelet aggregation.

Conclusions

Our results indicate that the CIRB site of TRPC6 plays an important functional role in platelets both modulating Ca2+ entry and aggregation through its interaction with CaM and IP3Rs.  相似文献   

10.
Glucagon induces intracellular Ca2+ ([Ca2+]i) elevation by stimulating glucagon receptor (GCGR). Such [Ca2+]i signaling plays important physiological roles, including glycogenolysis and glycolysis in liver cells and the survival of β-cells. Previous studies indicated that phospholipase C (PLC) might be involved in glucagon-mediated [Ca2+]i response. Other studies also debated whether cAMP accumulation mediated by GCGR/Gαs coupling contributes to [Ca2+]i elevation. But the exact mechanisms remain uncertain. In the present study, we found that glucagon induces [Ca2+]i elevation in HEK293 cells expressing GCGR. Removing extracellular Ca2+ did not affect glucagon-stimulated [Ca2+]i response. But depleting the intracellular Ca2+ store by thapsigargin completely inhibited glucagon-induced [Ca2+]i response. Experiments with forskolin and adenylyl cyclase inhibtor revealed that cAMP is not the cause of [Ca2+]i response. Further studies with Gαq/11 RNAi and pertussis toxin (PTX) indicated that both Gαq/11 and Gαi/o are involved. Combination of Gαq/11 RNAi and Gαi/o inhibition almost completely abolished glucagon-induced [Ca2+]i signaling.  相似文献   

11.
The objective of this study was to examine whether S-RNase plays a specific role in the pre-germinated Pyrus pollen. Effects of exogenous RNase and endogenous S-RNase on concentration of cytosolic-free calcium ([Ca2+]i) variation of pre-germinated Pyrus pollen were studied. [Ca2+]i variation caused by different RNases were complex. In 1 h after being cultured, exogenous RNase, RNase T1 and RNase A, and endogenous incompatible ‘Hohsui’ RNase promoted the [Ca2+]i of ‘Hohsui’ pollen. Acid proteins of ‘Hohsui’ had no remarkable influence on the [Ca2+]i of self-pollen. Endogenous compatible ‘Kohsui’ RNase reduced the [Ca2+]i of ‘Hohsui’ pollen, but compatible ‘Hohsui’ RNase can stimulate the [Ca2+]i of ‘Kohsui’ pollen. RNase T1, RNase A and incompatible ‘Kohsui’ S-RNase can also make ‘Kohsui’ pollen [Ca2+]i increase. Different from ‘Hohsui’ pollen, acid proteins of ‘Hohsui’ pull down the ‘Kohsui’ pollen [Ca2+]i remarkably. Conclusion can be made that during the prophase of pollen germination, endogenous S-RNase has no specific effect on pollen [Ca2+]i changes.  相似文献   

12.

Background/purpose

The goal of this study was to determine the role of the collagen binding receptor integrin α1β1 in regulating osmotically induced [Ca2+]i transients in chondrocytes.

Method

The [Ca2+]i transient response of chondrocytes to osmotic stress was measured using real-time confocal microscopy. Chondrocytes from wildtype and integrin α1-null mice were imaged ex vivo (in the cartilage of intact murine femora) and in vitro (isolated from the matrix, attached to glass coverslips). Immunocytochemistry was performed to detect the presence of the osmosensor, transient receptor potential vanilloid-4 (TRPV4), and the agonist GSK1016790A (GSK101) was used to test for its functionality on chondrocytes from wildtype and integrin α1-null mice.

Results/interpretation

Deletion of the integrin α1 subunit inhibited the ability of chondrocytes to respond to a hypo-osmotic stress with [Ca2+]i transients ex vivo and in vitro. The percentage of chondrocytes responding ex vivo was smaller than in vitro and of the cells that responded, more single [Ca2+]i transients were observed ex vivo compared to in vitro. Immunocytochemistry confirmed the presence of TRPV4 on wildtype and integrin α1-null chondrocytes, however application of GSK101 revealed that TRPV4 could be activated on wildtype but not integrin α1-null chondrocytes. Integrin α1β1 is a key participant in chondrocyte transduction of a hypo-osmotic stress. Furthermore, the mechanism by which integrin α1β1 influences osmotransduction is independent of matrix binding, but likely dependent on the chondrocyte osmosensor TRPV4.  相似文献   

13.
It is well established that intracellular calcium ([Ca2+]i) controls the inotropic state of the myocardium, and evidence mounts that a “Ca2+ clock” controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSCCa) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSCCa that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10 μM) and flufenamic acid (10 and 100 μM) decreases Ca2+ oscillations followed by an overall increase in [Ca2+]i. The latter occurs also in HL-1 cells in Ca2+-free solution and after depletion of sarcoplasmic reticulum Ca2+ with thapsigargin (10 μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130–150 kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca2+ oscillations followed by a compensatory increase in [Ca2+]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.  相似文献   

14.
Abstract

Protriptyline, a tricyclic anti-depressant, is used primarily to treat the combination of symptoms of anxiety and depression. However, the effect of protriptyline on prostate caner is unknown. This study examined whether the anti-depressant protriptyline altered Ca2+ movement and cell viability in PC3 human prostate cancer cells. The Ca2+-sensitive fluorescent dye fura-2 was used to measure [Ca2+]i. Protriptyline evoked [Ca2+]i rises concentration-dependently. The response was reduced by removing extracellular Ca2+. Protriptyline-evoked Ca2+ entry was inhibited by store-operated channel inhibitors (nifedipine, econazole and SKF96365), protein kinase C activator (phorbol 12-myristate 13 acetate, PMA) and protein kinase C inhibitor (GF109203X). Treatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-tert-butylhydr-oquinone (BHQ) in Ca2+-free medium inhibited 60% of protriptyline-evoked [Ca2+]i rises. Conversely, treatment with protriptyline abolished BHQ-evoked [Ca2+]i rises. Inhibition of phospholipase C with U73122 suppressed 50% of protriptyline-evoked [Ca2+]i rises. At concentrations of 50–70?µM, protriptyline decreased cell viability in a concentration-dependent manner; which were not reversed by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, in PC3 cells, protriptyline evoked [Ca2+]i rises by inducing phospholipase C-associated Ca2+ release from the endoplasmic reticulum and other stores, and Ca2+ influx via protein kinase C-sensitive store-operated Ca2+ channels. Protriptyline caused cell death that was independent of [Ca2+]i rises.  相似文献   

15.
We have recently documented that the Ca2+-permeable TRPV4 channel, which is abundantly expressed in distal nephron cells, mediates cellular Ca2+ responses to elevated luminal flow. In this study, we combined Fura-2-based [Ca2+]i imaging with immunofluorescence microscopy in isolated split-opened distal nephrons of C57BL/6 mice to probe the molecular determinants of TRPV4 activity and subcellular distribution. We found that activation of the PKC pathway with phorbol 12-myristate 13-acetate significantly increased [Ca2+]i responses to flow without affecting the subcellular distribution of TRPV4. Inhibition of PKC with bisindolylmaleimide I diminished cellular responses to elevated flow. In contrast, activation of the PKA pathway with forskolin did not affect TRPV4-mediated [Ca2+]i responses to flow but markedly shifted the subcellular distribution of the channel toward the apical membrane. These actions were blocked with the specific PKA inhibitor H-89. Concomitant activation of the PKA and PKC cascades additively enhanced the amplitude of flow-induced [Ca2+]i responses and greatly increased basal [Ca2+]i levels, indicating constitutive TRPV4 activation. This effect was precluded by the selective TRPV4 antagonist HC-067047. Therefore, the functional status of the TRPV4 channel in the distal nephron is regulated by two distinct signaling pathways. Although the PKA-dependent cascade promotes TRPV4 trafficking and translocation to the apical membrane, the PKC-dependent pathway increases the activity of the channel on the plasma membrane.  相似文献   

16.
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   

17.
Summary Intracellular calcium [Ca2+] i measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca2+] i transient followed by a steady-state increase as the characteristic [Ca2+] i response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca2+] i signal in single cells. The distribution of [Ca2+] i in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca2+] i present in the subplasmalemmal space and in one cell pole. [Ca2+] i gradients within these regions were not constant but showed temporal changes in the form of [Ca2+] i oscillations and spatial changes in the form of [Ca2+] i waves. [Ca2+] i oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca2+] and had a mean frequency of 12.6 +1.1 oscillations per min. The baseline [Ca2+], was 171 ± 13 nm and the mean oscillation amplitude was 194 ± 12 nm. Generation of [Ca2+] i waves was also independent of influx of extracellular Ca2+. [Ca2+] i waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +3 m per sec (n = 6). Increases of [Ca2+] i induced by different agonists were encoded into changes of baseline [Ca2+] i and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca2+] i regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca2+] i regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca2+] i oscillations.The authors thank Debbie Anderson for excellent technical assistance with the electron microscopy and Dr. M. Regoli for providing the NK-1 agonist [Sar9,Met(O2)11]-SP. This work was supported by National Institutes of Health Grants DK 40919 and DK 40675 and Veterans Administration Grant SMI.  相似文献   

18.
19.
External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca2+]i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca2+]i was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca2+]i in Jurkat T cells was 70 ± 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca2+]i to 52 ± 2 nM (n = 23), indicating that [Ca2+] entry from the external source is important for maintaining the basal level of [Ca2+]i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca2+]i by 30 ± 5% (P 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca2+]i responsiveness to EBE. Removal of external Ca2+ or Na+, but not Mg2+, inhibited the EBE-induced increase in [Ca2+]i. Dichlorobenzamil, an inhibitor of Na+/Ca2+ exchangers, also inhibited the EBE-induced increase in [Ca2+]i in a concentration-dependent manner with an IC50 of 0.11 ± 0.02 nM. When external [K+] was increased from 4.5 mM to 25 mM, EBE decreased [Ca2+]i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca2+ channel blocker. These results suggest that the EBE-induced [Ca2+]i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca2+]i is mediated by activation of Na+/Ca2+ exchangers and opening of L-type voltage-gated Ca2+ channels. (Mol Cell Biochem 271: 51–59, 2005)  相似文献   

20.
Summary The patch-clamp technique and measurements of single cell [Ca2+] i have been used to investigate the importance of extracellular Na+ for carbohydrate-induced stimulation of RINm5F insulin-secreting cells. Using patch-clamp whole-cell (current-clamp) recordings the average cellular transmembrane potential was estimated to be –60±1 mV (n=83) and the average basal [Ca2+] i 102±6nm (n=37). When challenged with either glucose (2.5–10mm) ord-glyceraldehyde (10mm) the cells depolarized, which led to the initiation of Ca2+ spike potentials and a sharp rise in [Ca2+] i . Similar effects were also observed with the sulphonylurea compound tolbutamide (0.01–0.1mm). Both the generation of the spike potentials and the increase in [Ca2+] i were abolished when Ca2+ was removed from the bathing media. When all external Na+ was replaced with N-methyl-d-glucamine, in the continued presence of either glucose,d-glyceraldehyde or tolbutamide, a membrane repolarization resulted, which terminated Ca2+ spike potentials and attenuated the rise in [Ca2+] i . Tetrodotoxin (TTX) (1–2 m) was also found to both repolarize the membrane and abolish secretagogue-induced rises in [Ca2+] i .  相似文献   

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