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1.
Effects of intracellular Mg2+ on a native Ca2+-and voltage-sensitive large-conductance K+ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode.
At an intracellular concentration of Ca2+ ([Ca2+]i) of 10−5–10−4 M, addition of 1–10 mM Mg2+ increased the open probability (Po) of the channel, which shifted the Po –membrane potential (Vm) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant).
However, the Mg2+-induced increase in Po was suppressed at a relatively low [Ca2+]i (10−5.5–10−6 M). Dwell-time histograms have revealed that addition of Mg2+ mainly increased Po by extending open times at 10−5 M Ca2+ and extending both open and closed times simultaneously at 10−5.5 M Ca2+. Since our data showed that raising the [Ca2+]i from 10−5 to 10−4 M increased Po mainly by shortening the closed time, extension of the closed time at 10−5.5 M Ca2+ would result from the Mg2+-inhibited Ca2+-dependent activation. At a constant Vm, adding Mg2+ enhanced the sigmoidicity of the Po–[Ca2+]i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg2+ on this channel is to elevate Po by lengthening the open time, while extension of the closed time at a relatively low [Ca2+]i results from a lowering of the sensitivity to Ca2+ of the channel by Mg2+, which causes the increase in the Hill coefficient.
M. Kubokawa and Y. Sohma contributed equally to this work. 相似文献
2.
The effect of ANG II on pHi, [Ca2+]i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo
4-AM and acridine orange, respectively. The recovery rate of pHi via the Na+/H+ exchanger was examined in the first 2 min following the acidification of pHi with a NH4Cl pulse. In the control situation, the pHi recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10−12 M or 10−9 M) increased this value (by 106% or 32%, respectively) but ANG II (10−7 M) decreased it to 47%. The control [Ca2+]i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late
and should not interfere in the measurements of pHi recovery and [Ca2+]i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent
calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate
that the biphasic effect of ANG II on Na+/H+ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway
activation (at 10−12 – 10−7 M ANG II) and by increases of [Ca2+]i in the lower range (at 10−12 M ANG II) and 2) inhibition of the exchanger at high [Ca2+]i levels (at 10−9 – 10−7 M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway. 相似文献
3.
E. A. Turovsky M. V. Turovskaya A. V. Berezhnov A. V. Tolmacheva N. P. Kaimachnikov L. P. Dolgacheva V. P. Zinchenko E. I. Maevskii V. V. Dynnik 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2012,6(1):35-44
Experiments on cultured mouse adipocytes (9 days in vitro) using fluorescent microscopy have shown that activation of α1- and α2-adrenoceptors by norepinephrine (NE) or α2-adrenoreceptors by L-arginine evokes transient Ca2+ signals, while activation of m3-cholinoreceptors by acetylcholine (ACh) or betaine causes sustained or damped Ca2+ oscillations. The presence in the incubation medium of L-arginine at a low concentration (100–200 μM) is necessary for a vigorous manifestation of these effects, apparently due to
transition of protein kinase G (PKG) and phosphodiesterase V into an active state. In the presence of 1–10 mM L-arginine, the amplitude of the Ca2+ transient response to NE increases and signal duration decreases. ACh and NE upon a sequential addition mutually potentiate
their effects. Using an inhibitory analysis we show that the observed modes are related to the operation of a signaling pathway
with the participation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), endothelial NO synthase (eNOS), cytoplasmic
guanylate cyclase (sGC), protein kinase G (PKG), ADP-ribosyl cyclase (CD38), and the ryanodine receptor (RyR). The formation
of several loops of positive feedbacks (PF) and negative feedbacks (NF) in the signaling system is possible: (i) short PF
loops due to Ca2+-induced Ca2+ release (CICR) from internal stores through the inositol trisphosphate receptor (IP3R) and RyR participating in the transient signal formation; (ii) long PF loop Ca2+ → eNOS → sGC → PKG → CD38 → RyR → Ca2+, which can provide necessary conditions for calcium oscillations arising from short PF loops (CICR); (iii) several NF loops
based on PKG-mediated inhibition of IP3R and activation of Ca2+-ATPases of sarco(endo)plasmic reticulum and of the plasma membrane providing a shutdown of signaling by the pathway phospholipase
C → IP3R → Ca2+ and limiting Ca2+ rise caused by the pathway PI3K → PKB → eNOS → sGC → PKG → CD38 → RyR → Ca2+. Convergence of signaling pathways that involve α1-, α2-, and m3-receptors and then Gβγ-subunits of Gq and Gq proteins acting on PI3Kγ can provide activation of cytoplasmic PKG, which plays a key role in producing transient responses,
in activation of Ca2+ removal and generation of [Ca2+]i oscillations. PKG inhibition (implemented here by KT5823 application) in the presence of any agonist results in rupture of
NF loops controlling Ca2+ transporting systems activity that leads to uncontrolled [Ca2+]i rise and cell death. 相似文献
4.
We evaluated mechanisms which mediate alterations in intracellular biochemical events in response to transient mechanical
stimulation of colonic smooth muscle cells. Cultured myocytes from the circular muscle layer of the rabbit distal colon responded
to brief focal mechanical deformation of the plasma membrane with a transient increase in intracellular calcium concentration
([Ca2+]
i
) with peak of 422.7 ± 43.8 nm above an average resting [Ca2+]
i
of 104.8 ± 10.9 nm (n= 57) followed by both rapid and prolonged recovery phases. The peak [Ca2+]
i
increase was reduced by 50% in the absence of extracellular Ca2+, while the prolonged [Ca2+]
i
recovery was either abolished or reduced to ≤15% of control values. In contrast, no significant effect of gadolinium chloride
(100 μm) or lanthanum chloride (25 μm) on either peak transient or prolonged [Ca2+]
i
recovery was observed. Pretreatment of cells with thapsigargin (1 μm) resulted in a 25% reduction of the mechanically induced peak [Ca2+]
i
response, while the phospholipase C inhibitor U-73122 had no effect on the [Ca2+]
i
transient peak. [Ca2+]
i
transients were abolished when cells previously treated with thapsigargin were mechanically stimulated in Ca2+-free solution, or when Ca2+ stores were depleted by thapsigargin in Ca2+-free solution. Pretreatment with the microfilament disrupting drug cytochalasin D (10 μm) or microinjection of myocytes with an intracellular saline resulted in complete inhibition of the transient. The effect
of cytochalasin D was reversible and did not prevent the [Ca2+]
i
increases in response to thapsigargin. These results suggest a communication, which may be mediated by direct mechanical
link via actin filaments, between the plasma membrane and an internal Ca2+ store.
Received: 24 March 1997/Revised: 21 July 1997 相似文献
5.
In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl− secretion to secretagogues acting via cAMP. Using a Ca2+ imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca2+]
i
via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase
in [Ca2+]
i
in normal (16HBE14o− cell line and primary lung culture) and in cystic fibrosis (CFTE29o− cell line) human airway epithelia. The potency order of nucleotides on [Ca2+]
i
variation was UTP ≫ ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca2+]
i
response could be mimicked by activation of CFTR with forskolin (20 μm) in a temperature-dependent manner. In 16HBE14o− cells, the forskolin-induced [Ca2+]
i
response increased with increasing temperature. In CFTE29o− cells, forskolin had no effect on [Ca2+]
i
at body temperature-forskolin-induced [Ca2+]
i
response in CF cells could only be observed at low experimental temperature (14°C) or when cells were cultured at 26°C instead
of 37°C. Pretreatment with CFTR channel blockers glibenclamide (100 μm) and DPC (100 μm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 μm), inhibited the forskolin [Ca2+]
i
response. Together, these results demonstrate that once activated, CFTR regulates [Ca2+]
i
by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia.
Received: 3 April 2000/Revised: 30 June 2000 相似文献
6.
Craviso GL Choe S Chatterjee P Chatterjee I Vernier PT 《Cellular and molecular neurobiology》2010,30(8):1259-1265
Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca2+ entry into the cells via L-type voltage-gated Ca2+ channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond
pulse-induced effects on intracellular Ca2+ level ([Ca2+]i) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca2+ entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude
of the rise in [Ca2+]i elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and
P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca2+]i response of the cells, suggesting Ca2+ influx occurs only via VGCCs. Lowering extracellular K+ concentration from 5 to 2 mM or pulsing cells in Na+-free medium suppressed the pulse-induced rise in [Ca2+]i in the majority of cells. Thus, both membrane potential and Na+ entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca2+ influx. However, activation of voltage-gated Na+ channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca2+]i. These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple
types of VGCCs in chromaffin cells in a manner involving Na+ transport across the plasma membrane. Whether Na+ transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined. 相似文献
7.
Summary Discrepancies about the role of L-type voltage-gated calcium channels (VGCC) in acetylcholine (ACh)-induced [Ca2+]i oscillations in tracheal smooth muscle cells (TSMCs) have been seen in recent reports. We demonstrate here that ACh-induced
[Ca2+]i oscillations in TMCS were reversibly inhibited by three VGCC blockers, nicardipine, nifedipine and verapamil. Prolonged (several
minutes) application of VGCC blockers, led to tachyphylaxis; that is, [Ca2+]i oscillations resumed, but at a lower frequency. Brief (15–30 s) removal of VGCC blockers re-sensitized [Ca2+]i oscillations to inhibition by the agents. Calcium oscillations tolerant to VGCC blockers were abolished by KB-R7943, an inhibitor
of the reverse mode of Na+/Ca2+ exchanger (NCX). KB-R7943 alone also abolished ACh-induced [Ca2+]i oscillations. Enhancement of the reverse mode of NCX via removing extracellular Na+ reversed inhibition of ACh-induced [Ca2+]i oscillations by VGCC blockers. Inhibition of non-selective cation channels using Gd3+ slightly reduced the frequency of ACh-induced [Ca2+]i oscillations, but did not prevent the occurrence of tachyphylaxis. Altogether, these results suggest that VGCC and the reverse
mode of NCX are two primary Ca2+ entry pathways for maintaining ACh-induced [Ca2+]i oscillations in TSMCs. The two pathways complement each other, and may account for tachyphylaxis of ACh-induced [Ca2+]i oscillations to VGCC blockers. 相似文献
8.
O. Strauß K. Steinhausen S. Mergler F. Stumpff M. Wiederholt 《The Journal of membrane biology》1999,169(3):141-153
This combined study of patch-clamp and intracellular Ca2+ ([Ca2+]
i
) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca2+-dependent Cl− channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca2+]
i
and activation of Cl− currents. In contrast, intracellular application of Ca2+ (10 μm) only induced transient activation of Cl− currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca2+ and to the blocker of I
CRAC, La3+ (10 μm), despite the fact that both maneuvers led to a decline in [Ca2+]
i
. The InsP3-induced rise in Cl− conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca2+ stores or by removal of Ca2+ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca2+-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca2+/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl−-conductance in 50% of the cells investigated without affecting [Ca2+]
i
. Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca2+]
i
and Cl− conductance. In summary, elevation of [Ca]
i
by InsP3 leads to activation of Cl− channels involving cytosolic Ca2+ stores and Ca2+ influx from extracellular space. Tyrosine kinases are essential for the Ca2+-independent maintenance of this conductance.
Received: 15 October 1998/Revised: 3 March 1999 相似文献
9.
In cystic fibrosis, the mutation of the CFTR protein causes reduced transepithelial Cl− secretion. As recently proposed, beside its role of Cl− channel, CFTR may regulate the activity of other channels such as a Ca2+-activated Cl− channel. Using a calcium imaging system, we show, in adenovirus-CFTR infected Chinese Hamster Ovary (CHO) cell monolayers,
that CFTR can act as a regulator of intracellular [Ca2+]
i
([Ca2+]
i
), involving purino-receptors. Apical exposure to ATP or UTP produced an increase in ([Ca2+]
i
in noninfected CHO cell monolayers (CHO-WT), in CHO monolayers infected with an adenovirus-CFTR (CHO-CFTR) or infected with
an adenovirus-LacZ (CHO-LacZ). The transient [Ca2+]
i
increase produced by ATP or UTP could be mimicked by activation of CFTR with forskolin (20 μm) in CHO-CFTR confluent monolayers. However, forskolin had no significant effect on [Ca2+]
i
in noninfected CHO-WT or in CHO-LacZ cells. Pretreatment with purino-receptor antagonists such as suramin (100 μm) or reactive blue-2. (100 μm), and with hexokinase (0.28 U/mg) inhibited the [Ca2+]
i
response to forskolin in CHO-CFTR infected cells. Taken together, our experiments provide evidence for purino-receptor activation
by ATP released from the cell and regulation of [Ca2+]
i
by CFTR in CHO epithelial cell membranes.
Received: 5 April 1999/Revised: 28 June 1999 相似文献
10.
The actions of the neonicotinoid imidacloprid on cholinergic neurons of Drosophila melanogaster 总被引:2,自引:0,他引:2
The neonicotinoid insecticide imidacloprid is an agonist on insect nicotinic acetylcholine receptors (nAChRs). We utilised
fura-2-based calcium imaging to investigate the actions of imidacloprid on cultured GFP-tagged cholinergic neurons from the
third instar larvae of the genetic model organism Drosophila melanogaster. We demonstrate dose-dependent increases in intracellular calcium ([Ca2+]i) in cholinergic neurons upon application of imidacloprid (10 nM–100 μM) that are blocked by nAChR antagonists mecamylamine
(10 μM) and α-bungarotoxin (α-BTX, 1 μM). When compared to other (untagged) neurons, cholinergic neurons respond to lower
concentrations of imidacloprid (10–100 nM) and exhibit larger amplitude responses to higher (1–100 μM) concentrations of imidacloprid.
Although imidacloprid acts via nAChRs, increases in [Ca2+]i also involve voltage-gated calcium channels (VGCCs) in both groups of neurons. Thus, we demonstrate that cholinergic neurons
express nAChRs that are highly sensitive to imidacloprid, and demonstrate a role for VGCCs in amplifying imidacloprid-induced
increases in [Ca2+]i. 相似文献
11.
We investigated the contribution of L-, N- and P/Q-type Ca2+ channels to the [Ca2+]i changes, evoked by kainate, in the cell bodies of hippocampal neurons, using a pharmacological approach and Ca2+ imaging. Selective Ca2+ channel blockers, namely nitrendipine, ω-Conotoxin GVIA (ω-GVIA) and ω-Agatoxin IVA (ω-AgaIVA) were used. The [Ca2+]i changes evoked by kainate presented a high variability, and were abolished by NBQX, a AMPA/kainate receptor antagonist, but
the N-methyl-d-aspartate (NMDA) receptor antagonist, D-AP5, was without effect. Each Ca2+ channel blocker caused differential inhibitory effects on [Ca2+]i responses evoked by kainate. We grouped the neurons for each blocker in three subpopulations: (1) neurons with responses
below 60% of the control; (2) neurons with responses between 60% and 90% of the control, and (3) neurons with responses above
90% of the control. The inhibition caused by nitrendipine was higher than the inhibition caused by ω-GVIA or ω-AgaIVA. Thus,
in the presence of nitrendipine, the percentage of cells with responses below 60% of the control was 41%, whereas in the case
of ω-GVIA or ω-AgaIVA the values were 9 or 17%, respectively. The results indicate that hippocampal neurons differ in what
concerns their L-, N- and P/Q- type Ca2+ channels activated by stimulation of the AMPA/kainate receptors.
Special issue article in honor of Dr. Ricardo Tapia. 相似文献
12.
Preload-induced changes of active tension and [Ca2+]i are “dissociated” in mammalian myocardium. This study aimed to describe the distinct effects of preload at low and physiological
[Ca2+]o. Rat RV papillary muscles were studied in isometric conditions at 25‡C and 0.33 Hz at 1 mM (hypo-Ca group) and 2.5 mM [Ca2+]o (normal-Ca group). [Ca2+]i was monitored with fura-2/AM. Increase of preload caused a rise of active tension in hypo-Ca and normal-Ca groups whereas
peak fluorescence rose significantly only at low [Ca2+]o. End-diastolic tension, end-diastolic level of fluorescence, time-to-peak tension, but not time-to-peak of Ca2+ transient, progressively increased with preload. Mechanical relaxation decelerated with preload while Ca2+ transient decay time decreased in the initial phase and increased in the late phase, resulting in a prominent “bump” configuration.
The “bump” was assessed as a ratio of its area to the fluorescence trace area. It was a new finding that the preload-induced
rise of this ratio was twice as large in hypo-Ca. Our results indicate that preload-induced changes in active tension and
[Ca2+]i are “dissociated” in rat myocardium, with relatively higher expression at low [Ca2+]o. Ca-dependence of Ca-TnC association/dissociation kinetics is thought to be a main contributor to these preload-induced effects. 相似文献
13.
Schaloske RH Lusche DF Bezares-Roder K Happle K Malchow D Schlatterer C 《BMC cell biology》2005,6(1):13
Background
Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i-change is composed of liberation of stored Ca2+ and extracellular Ca2+-entry. The significance of the [Ca2+]i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca2+-buffers in the cytosol indicates that a [Ca2+]i-increase is required for chemotaxis. Yet, the iplA - mutant disrupted in a gene bearing similarity to IP3-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca2+]i-transient which favours the view that [Ca2+]i-changes are insignificant for chemotaxis. 相似文献14.
E. A. Turovskii M. V. Konakov A. V. Berezhnov V. P. Zinchenko G. E. Bronnikov L. P. Dolgacheva 《Cell and Tissue Biology》2011,5(5):511-519
The thermogenic capability of brown adipose tissue is controlled by noradrenaline. By interacting with α1- and β-adrenoreceptors
of adipocytes, noradrenaline (NA) increases the intracellular concentration of Ca2+ ([Ca2+]i) and cAMP. The changes in [Ca2+]i under the action of NA and selective agonists of α1- and β-adrenoreceptors, i.e., cirazoline and isoproterenol (IP), are
recorded on individual cells of the primary culture of adipocytes during the day in vitro (DIV) 1, DIV 3, and DIV 6. The change
in [Ca2+]i under the effect of IP as compared to the response to cirazoline in cells of DIV 1 is characterized by a higher amplitude
and shorter duration of impulses in the entire diapason of the used physiological concentrations. After DIV 3, these differences
are insignificant and, after DIV 6, the differences in kinetics are nearly absent. For all three agonists, the kinetics of
the [Ca2+]i change in the proliferating and differentiated cells is significantly different; i.e., the response amplitude increases with
the age of the culture and the duration of transitory response decreases, while sensitivity to agonists of adrenoreceptors
increases. It can be seen from the rise in [Ca2+]i with an inhibitor of Ca2+-ATPase of the endoplasmic reticulum thapsigargin in calcium-free medium that the source of calcium ions in the endoplasmic
reticulum rises with the growth and development of cells in culture, while the rate at which Ca2+ is pumped out of cells, which characterizes the activity of Ca2+-ATPase of the plasma membrane, increases. 相似文献
15.
The objective of this study was to investigate the influences of carbonyl stress induced by malondialdehyde (MDA), a typical
intermediate of lipid peroxidation, on intracellular free Ca2+ concentration ([Ca2+]i) alterations in cultured hippocampal neurons of rat. The microphotographic study clearly demonstrated that the hippocampal
neurons became gradually damaged following exposure to different concentrations of MDA. Further study indicated that the plasma
membrane Ca2+-ATPase (PMCA) activity was inhibited by MDA in a concentration- and time-dependent manner. The supplementation of 100 μM
MDA was found to cause a notable early phase increase of [Ca2+]i in hippocampal neuron cultures followed by a more pronounced late-phase elevation of [Ca2+]i. Such effect of MDA was prevented by the addition of nimodipine, an inhibitor of L-type calcium channel or by an extracellular
Ca2+ chelator EGTA. The identification of the calcium signalling pathways were studied by applying U73122, an inhibitor of PL-C,
and H-89, an inhibitor of protein kinase A (PKA), showing the involvement of PL-C/IP3 pathway but not the PKA/cAMP pathway.
These results suggested that MDA-related carbonyl stress caused damages of rat hippocampal neurons by triggering Ca2+ influx and influencing Ca2+ homeostasis in cultured neurons, and also MDA may act as a signalling molecule regulating Ca2+ release from intracellular stores. 相似文献
16.
M.-P. Blanchard N. Klauke S. Zitzmann H. Plattner 《The Journal of membrane biology》1999,169(3):155-165
We analyzed [Ca2+]
i
transients in Paramecium cells in response to veratridine for which we had previously established an agonist effect for trichocyst exocytosis (Erxleben
& Plattner, 1994. J. Cell Biol.
127:935–945; Plattner et al., 1994. J. Membrane Biol.
158:197–208). Wild-type cells (7S), nondischarge strain nd9–28°C and trichocyst-free strain ``trichless' (tl), respectively,
displayed similar, though somewhat diverging time course and plateau values of [Ca2+]
i
transients with moderate [Ca2+]
o
in the culture/assay fluid (50 μm or 1 mm). In 7S cells which are representative for a normal reaction, at [Ca2+]
o
= 30 nm (c.f. [Ca2+]
rest
i
=∼50 to 100 nm), veratridine produced only a small cortical [Ca2+]
i
transient. This increased in size and spatial distribution at [Ca2+]
o
= 50 μm of 1 mm. Interestingly with unusually high yet nontoxic [Ca2+]
o
= 10 mm, [Ca2+]
i
transients were much delayed and also reduced, as is trichocyst exocytosis. We interpret our results as follows. (i) With
[Ca2+]
o
= 30 nm, the restricted residual response observed is due to Ca2+ mobilization from subplasmalemmal stores. (ii) With moderate [Ca2+]
o
= 50 μm to 1 mm, the established membrane labilizing effect of veratridine may activate not only subplasmalemmal stores but also Ca2+
o influx from the medium via so far unidentified (anteriorly enriched) channels. Visibility of these phenomena is best in tl
cells, where free docking sites allow for rapid Ca2+ spread, and least in 7S cells, whose perfectly assembled docking sites may ``consume' a large part of the [Ca2+]
i
increase. (iii) With unusually high [Ca2+]
o
, mobilization of cortical stores and/or Ca2+
o
influx may be impeded by the known membrane stabilizing effect of Ca2+
o
counteracting the labilizing/channel activating effect of veratridine. (iv) We show these effects to be reversible, and,
hence, not to be toxic side-effects, as confirmed by retention of injected calcein.
(v) Finally, Mn2+ entry during veratridine stimulation, documented by Fura-2 fluorescence quenching, may indicate activation of unspecific
Me2+ channels by veratridine. Our data have some bearing on analysis of other cells, notably neurons, whose response to veratridine
is of particular and continous interest.
Received: 8 December 1998/Revised: 2 March 1999 相似文献
17.
P. K. Aley C. C. Bauer M. L. Dallas J. P. Boyle K. E. Porter C. Peers 《The Journal of membrane biology》2009,227(3):151-158
Our understanding of vascular endothelial cell physiology is based on studies of endothelial cells cultured from various vascular
beds of different species for varying periods of time. Systematic analysis of the properties of endothelial cells from different
parts of the vasculature is lacking. Here, we compare Ca2+ homeostasis in primary cultures of endothelial cells from human internal mammary artery and saphenous vein and how this is
modified by hypoxia, an inevitable consequence of bypass grafting (2.5% O2, 24 h). Basal [Ca2+]
i
and store depletion-mediated Ca2+ entry were significantly different between the two cell types, yet agonist (ATP)–mediated mobilization from endoplasmic reticulum
stores was similar. Hypoxia potentiated agonist-evoked responses in arterial, but not venous, cells but augmented store depletion-mediated
Ca2+ entry only in venous cells. Clearly, Ca2+ signaling and its remodeling by hypoxia are strikingly different in arterial vs. venous endothelial cells. Our data have
important implications for the interpretation of data obtained from endothelial cells of varying sources. 相似文献
18.
Long LH Liu J Liu RL Wang F Hu ZL Xie N Fu H Chen JG 《Cellular and molecular neurobiology》2009,29(1):7-15
Methionine and cysteine residues in proteins are the major targets of reactive oxygen species (ROS). The present work was
designed to characterize the impact of methionine and cysteine oxidation upon [Ca2+]i in hippocampal neurons. We investigated the effects of H2O2 and chloramine T(Ch-T) agents known to oxidize both cysteine and methionine residues, and 5, 5′-dithio-bis (2-nitrobenzoic
acid) (DTNB)—a cysteine-specific oxidant, on the intracellular calcium in hippocampal neurons. The results showed that these
three oxidants, 1 mM H2O2, 1 mM Ch-T, and 500 μM DTNB, induced an sustained elevation of [Ca2+]i by 76.1 ± 3.9%, 86.5 ± 5.0%, and 24.4 ± 3.2% over the basal level, respectively. The elevation induced by H2O2 and Ch-T was significantly higher than DTNB. Pretreatment with reductant DTT at 1 mM for 10 min completely prevented the
action of DTNB on [Ca2+]i, but only partially reduced the effects of H2O2 and Ch-T on [Ca2+]i, the reductions were 44.6 ± 4.2% and 29.6 ± 6.1% over baseline, respectively. The elevation of [Ca2+]i induced by H2O2 and Ch-T after pretreatment with DTT were statistically higher than that induced by single administration of DTNB. Further
investigation showed that the elevation of [Ca2+]i mainly resulted from internal calcium stores. From our data, we propose that methionine oxidation plays an important role
in the regulation of intracellular calcium and this regulation may mainly be due to internal calcium stores. 相似文献
19.
Lahcen Iouzalen Frdrique Lantoine Marie-Gabrielle Pernollet Elisabeth Millanvoye-Van Brussel Marie-Aude Devynck Monique David-Dufilho 《Cell calcium》1996,20(6):501-508
The effects of the imidazole compound SK&F 96365 on Ca2+ movements and production of nitric oxide (NO) and von Willebrand factor (vWF) have been investigated in human endothelial cells. Changes in cytosolic Ca2+ concentration ([Ca2+]i) were measured with Fura-2. Real-time production of NO was monitored with a porphyrinic microsensor and the release of vWF with an enzyme-linked immunosorbent assay. Irrespective of the transmembrane Ca2+ gradient, 30 μM SK&F 96365 doubled [Ca2+]i suggesting a Ca2+ release from intracellular stores. The SK&F 96365-induced [Ca2+]i rise was not accompanied by detectable NO and vWF production, while 1 μM thapsigargin enhanced [Ca2+]i 2.5 times, doubled the secretion of vWF and increased the NO production to 10 ± 4 nM (n = 5). Pretreatment with SK&F 96365 prevented thapsigargin from increasing [Ca2+]i, NO production and vWF secretion. To investigate the mechanism by which SK&F 96365 released Ca2+, from internal pools, its effect and that of thapsigargin on the ATP-dependent 45Ca2+, uptake into platelet membrane vesicles were compared. SK&F 96365 as thapsigargin, dose-dependently reduced the initial rate of 45Ca2+ uptake. In conclusion, we demonstrate that, in the absence of Ca2+ entry from the extracellular space, the [Ca2+]i increase elicited by SK&F 96365 or thapsigargin is not sufficient to initiate NO synthesis and vWF secretion. This confirms the important role of Ca2+ influx in endothelial secretion processes. 相似文献
20.
Xiaohong Liu Junwei Zeng Yandong Zhao Zhi Xiao Chuanqing Fang Huaizhen Ruan 《Neurochemical research》2010,35(5):804-810
In addition to the classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomic effects on neurons.
In the present study, the effect of corticosterone (CORT) on ATP-induced Ca2+ mobilization in cultured dorsal root ganglion (DRG) neurons were detected with confocal laser scanning microscopy using fluo-4/AM
as a calcium fluorescent indicator that could monitor real-time alterations of intracellular calcium concentration ([Ca2+]i). ATP, an algesic agent, caused [Ca2+]i increase in DRG neurons by activation of P2X receptor. Pretreatment with CORT (1 nM–1 μM for 5 min) inhibited ATP-induced
[Ca2+]i increase in DRG neurons. The rapid inhibition of ATP-induced Ca2+ response by CORT was concentration-dependent, reversible and could be blocked by glucocorticoid receptor antagonist RU38486
(10 μM). Furthermore, the inhibitory effect of CORT was abolished by protein kinase A inhibitor H89 (10 μM), but was not influenced
by protein kinase C inhibitor Chelerythrine chloride (10 μM). On the other hand, membrane-impermeable bovine serum albumin-conjugated
corticosterone had no effect on ATP-induced [Ca2+]i transients. These observations suggest that a nongenomic pathways may be involved in the effect of CORT on ATP-induced
[Ca2+]i transients in cultured DRG neurons. 相似文献