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1.
Summary Neuropeptide tachykinins, present within sensory nerves, have been implicated as neurotransmitters involved in nonadrenergic and noncholinergic airway muscle contraction. The signal transduction pathways of tachykinins on muscle contraction and Ca2+ mobilization were investigated in swine trachea. Tachykinins, substance P (SP) and neurokinin A (NKA), concentration (1 nM to 1 μM)-dependently induced contractile responses with removal of epithelium, whereas neurokinin B (NKB) did not alter the muscle tension. The SP- and NKA-evoked muscle contractions were inhibited by NK1-R antagonist L732138, but not by either NK2-R antagonist MDL29913 or NK3-R antagonist SB218795. Consistently, SP-elicited increase in [Ca2+]i was abolished by NK1-R antagonist, neither by NK2-R nor NK3-R antagonists. The SP-induced muscular responses were significantly inhibited by L-type Ca2+ channel blocker verapamil and withdrawal of external Ca2+. Caffeine (10 mM) or ryanodine (50 μM) also partly suppressed the SP-induced muscle responses. Inhibition of inositol 1,4,5-trisphosphate (InsP3) receptor with 2-APB (75 μM) potently attenuated SP-evoked Ca2+ mobilization and muscle contraction, which was further inhibited by 2-APB under Ca2+-free external solution, but not completely. Unexpectedly, simultaneous blockade of InsP3 receptor and ryanodine receptor (RyR) by 2-APB and ryanodine enhanced SP-evoked muscle contraction and Ca2+ mobilization. This potentiation was virtually abolished by removal of external Ca2+, suggesting native Ca2+ channels may contribute to this phenomenon. These results demonstrate that tachykinins produce a potent muscle contraction associated with Ca2+ mobilization via tachykinin NK1- R-dependent activation of multiple signal transduction pathways involving Ca2+ influx and release of Ca2+ from InsP3- and ryanodine-sensitive Ca2+ stores. Blockade of both InsP3 receptor and RyR enhances the Ca2+ influx through native Ca2+ channels in plasma membrane, which is crucial to Ca2+ signaling in response to NK1 receptor activation. 相似文献
2.
Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca2+]i) and insulin release in mouse pancreatic β-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6
insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca2+]i rise via cytoplasmic Ca2+ mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 μM) transiently increased [Ca2+]i in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y1 receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM),
whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y6 receptor agonist UDP (200 μM) transiently increased [Ca2+]i and reduced insulin secretion at high glucose, whereas the P2Y2/4 receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca2+]i transients induced by 2-MeSADP and UDP were antagonized by suramin (100 μM), U73122 (2 μM, PLC inhibitor), and 2-APB (10
or 30 μM, IP3 receptor antagonist), but neither by staurosporine (1 μM, PKC inhibitor) nor depletion of extracellular Ca2+. The effect of 2-MeSADP on [Ca2+]i was also significantly inhibited by MRS2500, a P2Y1 receptor antagonist. These results suggested that P2Y1 and P2Y6 receptor subtypes are involved in Ca2+ mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP
transiently elevated [Ca2+]i and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs
of P2Y1 and P2Y6, but not P2Y2 and P2Y4 receptors. 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.
S. Pedersen E.K. Hoffmann C. Hougaard N.K. Jørgensen G.B. Wybrandt I.H. Lambert 《The Journal of membrane biology》1997,155(1):61-73
Stimulation of Ehrlich ascites tumor cells with leukotriene D4 (LTD4) within the concentration range 1–100 nm leads to a concentration-dependent, transient increase in the intracellular, free Ca2+ concentration, [Ca2+]
i
. The Ca2+ peak time, i.e., the time between addition of LTD4 and the highest measured [Ca2+]
i
value, is in the range 0.20 to 0.21 min in ten out of fourteen independent experiments. After addition of a saturating concentration
of LTD4 (100 nm), the highest measured increase in [Ca2+]
i
in Ehrlich cells suspended in Ca2+-containing medium is 260 ± 14 nm and the EC50 value for LTD4-induced Ca2+ mobilization is estimated at 10 nm. Neither the peptido-leukotrienes LTC4 and LTE4 nor LTB4 are able to mimic or block the LTD4-induced Ca2+ mobilization, hence the receptor is specific for LTD4. Removal of Ca2+ from the experimental buffer significantly reduces the size of the LTD4-induced increase in [Ca2+]
i
. Furthermore, depletion of the intracellular Ins(1,4,5)P3-sensitive Ca2+ stores by addition of the ER-Ca2+-ATPase inhibitor thapsigargin also reduces the size of the LTD4-induced increase in [Ca2+]
i
in Ehrlich cells suspended in Ca2+-containing medium, and completely abolishes the LTD4-induced increase in [Ca2+]
i
in Ehrlich cells suspended in Ca2+-free medium containing EGTA. Thus, the LTD4-induced increase in [Ca2+]
i
in Ehrlich cells involves an influx of Ca2+ from the extracellular compartment as well as a release of Ca2+ from intracellular Ins(1,4,5)P3-sensitive stores. The Ca2+ peak times for the LTD4-induced Ca2+ influx and for the LTD4-induced Ca2+ release are recorded in the time range 0.20 to 0.21 min in four out of five experiments and in the time range 0.34 to 0.35
min in six out of eight experiments, respectively. Stimulation with LTD4 also induces a transient increase in Ins(1,4,5)P3 generation in the Ehrlich cells, and the Ins(1,4,5)P3 peak time is recorded in the time range 0.27 to 0.30 min. Thus, the Ins(1,4,5)P3 content seems to increase before the LTD4-induced Ca2+ release from the intracellular stores but after the LTD4-induced Ca2+ influx. Inhibition of phospholipase C by preincubation with U73122 abolishes the LTD4-induced increase in Ins(1,4,5)P3 as well as the LTD4-induced increase in [Ca2+]
i
, indicating that a U73122-sensitive phospholipase C is involved in the LTD4-induced Ca2+ mobilization in Ehrlich cells. The LTD4-induced Ca2+ influx is insensitive to verapamil, gadolinium and SK&F 96365, suggesting that the LTD4-activated Ca2+ channel in Ehrlich cells is neither voltage gated nor stretch activated and most probably not receptor operated. In conclusion,
LTD4 acts in the Ehrlich cells via a specific receptor for LTD4, which upon stimulation initiates an influx of Ca2+, through yet unidentified Ca2+ channels, and an activation of a U73122-sensitive phospholipase C, Ins(1,4,5)P3 formation and finally release of Ca2+ from the intracellular Ins(1,4,5)P3-sensitive stores.
Received: 9 February 1996/Revised: 15 August 1996 相似文献
5.
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
6.
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
7.
Liu J Wang F Huang C Long LH Wu WN Cai F Wang JH Ma LQ Chen JG 《Cellular and molecular neurobiology》2009,29(3):317-328
Recent evidences indicate the existence of an atypical D1 dopamine receptor other than traditional D1 dopamine receptor in the brain that mediates PI hydrolysis via activation of phospholipase Cβ (PLCβ). To further understand the basic physiological function of this receptor in brain, the effects of a selective phosphoinositide
(PI)-linked D1 dopamine receptor agonist SKF83959 on cytosolic free calcium concentration ([Ca2+]i) in cultured rat prefrontal cortical astrocytes were investigated by calcium imaging. The results indicated that SKF83959
caused a transient dose-dependent increase in [Ca2+]i. Application of D1 receptor, but not D2, α1 adrenergic, 5-HT receptor, or cholinergic antagonist prevented SKF83959-induced [Ca2+]i rise, indicating that activation of the D1 dopamine receptor was essential for this response. Increase in [Ca2+]i was a two-step process characterized by an initial increase in [Ca2+]i mediated by release from intracellular stores, supplemented by influx through voltage-gated calcium channels, receptor-operated
calcium channels, and capacitative Ca2+ entry. Furthermore, SKF83959-stimulated increase in [Ca2+]i was abolished following treatment with a PLC inhibitor. Overall, these results suggested that activation of D1 receptor by SKF83959 mediates a dose-dependent mobilization of [Ca2+]i via the PLC signaling pathway in cultured rat prefrontal cortical astrocytes. 相似文献
8.
Michael ter Braak Kerstin Danneberg Karin Lichte Kerstin Liphardt Nicholas T. Ktistakis Stuart M. Pitson Timothy Hla Karl H. Jakobs Dagmar Meyer zu Heringdorf 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2009,1791(5):357-370
Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of Gq-coupled receptors induced a profound, rapid (half-life 3–5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein α-subunits specifically of the Gq family. Classical Gq signalling pathways, or phosphorylation at Ser225, phospholipase D and Ca2+/calmodulin were not involved in M3 receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of Gq-coupled receptors, linking them via cross-activation of S1P receptors to Gi and G12/13 signalling pathways. 相似文献
9.
Reshkin SJ Guerra L Bagorda A Debellis L Cardone R Li AH Jacobson KA Casavola V 《The Journal of membrane biology》2000,178(2):103-113
We have previously demonstrated that in A6 renal epithelial cells, a commonly used model of the mammalian distal section of the nephron, adenosine A1 and A2A receptor activation modulates sodium and chloride transport and intracellular pH (Casavola et al., 1997). Here we show that
apical addition of the A3 receptor-selective agonist, 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-methyluronamide (Cl-IB-MECA) stimulated a chloride secretion that was mediated by calcium- and
cAMP-regulated channels. Moreover, in single cell measurements using the fluorescent dye Fura 2-AM, Cl-IB-MECA caused an increase
in Ca2+ influx. The agonist-induced rise in [Ca2+]
i
was significantly inhibited by the selective adenosine A3 receptor antagonists, 2,3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS 1523) and 3-ethyl 5-benzyl
2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS 1191) but not by antagonists of either A1 or A2 receptors supporting the hypothesis that Cl-IB-MECA increases [Ca2+]
i
by interacting exclusively with A3 receptors. Cl-IB-MECA-elicited Ca2+ entry was not significantly inhibited by pertussis toxin pretreatment while being stimulated by cholera toxin preincubation
or by raising cellular cAMP levels with forskolin or rolipram. Preincubation with the protein kinase A inhibitor, H89, blunted
the Cl-IB-MECA-elicited [Ca2+]
i
response. Moreover, Cl-IB-MECA elicited an increase in cAMP production that was inhibited only by an A3 receptor antagonist. Altogether, these data suggest that in A6 cells a G
s
/protein kinase A pathway is involved in the A3 receptor-dependent increase in calcium entry.
Received: 9 March 2000/Revised: 14 August 2000 相似文献
10.
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
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.
R. Leurs E. Traiffort J. M. Arrang J. Tardivel-Lacombe M. Ruat J.-C. Schwartz 《Journal of neurochemistry》1994,62(2):519-527
Abstract: A cDNA encoding a guinea pig histamine H1 receptor was stably expressed in Chinese hamster ovary (CHO) cells. In one resulting clone, named CHO(H1), the H1 receptor was found to be coupled to several major signal transduction pathways. In each case the involvement of a Gi/Go protein with pertussis toxin (PTX) was assessed, as well as the influence of extracellular Ca2+ and of protein kinase C activation by phorbol 12-myristate 13-acetate (PMA). Histamine induced, in a PTX- and PMA-insensitive manner, a biphasic increase in the intracellular Ca2+ level of which only the second sustained phase was dependent on the extracellular Ca2+ level. Histamine also caused a threefold elevation of inositol phosphate production, which was PTX-insensitive, but slightly inhibited by PMA and reduced by 75% in the absence of extracellular Ca2+. Histamine also caused a massive release of arachidonic acid, which occurred in a Ca2+- and PMA-sensitive manner, probably through the activation of a cytosolic phospholipase A2, which partly involves coupling to a PTX-sensitive G protein. In comparison, in HeLa cells endowed with a native H1 receptor, the histamine-induced arachidonic acid release was also Ca2+- and PMA-sensitive, but totally PTX-insensitive. Finally, in CHO(H1) cells, histamine in very low concentrations potentiated the cyclic AMP accumulation induced by forskolin. This response appeared to be insensitive to PTX, extracellular Ca2+, and PMA. These various observations show that stimulation of a single receptor subtype, the guinea pig H1 receptor, can trigger four major intracellular signals through coupling to several G proteins that are variously modulated by extracellular Ca2+ and protein kinase C activation. 相似文献
13.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1997,1355(1):89-98
A cDNA encoding a mouse B2 bradykinin (BK) receptor was stably transfected in Chinese hamster ovary (CHO) cells. In two resulting transformants, mouse B2 BK receptor was found to induce a twofold elevation in the inositol-1,4,5-trisphosphate level. In a pertussis toxin-insensitive manner, BK also produced a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). The initial elevation in [Ca2+]i was abolished by thapsigargin pretreatment in Ca2+-free medium. The second phase was dependent on external Ca2+. The BK/inositol trisphosphate- and thapsigargin-sensitive Ca2+ stores required extracellular Ca2+ for refilling. Ca2+ influx induced by BK and thapsigargin was confirmed by Mn2+ entry through Ca2+ influx pathways producing Mn2+ quenching. Genistein, a tyrosine kinase inhibitor, partially decreased the BK-induced [Ca2+]i increase during the sustained phase and the rate of Mn2+ entry. BK had essentially no effect on the intracellular cyclic AMP level. The results suggest that the mouse B2 BK receptor couples to phospholipase C in CHO cells and that its activation results in biphasic [Ca2+]i increases, by mobilization of intracellular Ca2+ and store-depletion-mediated Ca2+ influx, the latter of which is tyrosine phosphorylation-dependent. 相似文献
14.
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
15.
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
16.
17.
Caffeine causes a [Ca2+]
i
increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca2+]rest
i
∼50 to 80 nm, [Ca2+]act
i
rises within ≤3 sec to 500 (trichocyst-free strain tl) or 220 nm (nondischarge strain nd9–28°C) in the cortex. Rapid confocal analysis of wildtype cells (7S) showed only a 2-fold cortical
increase within 2 sec, accompanied by trichocyst exocytosis and a central Ca2+ spread during the subsequent ≥2 sec. Chelation of Ca2+
o
considerably attenuated [Ca2+]
i
increase. Therefore, caffeine may primarily mobilize cortical Ca2+ pools, superimposed by Ca2+ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst
contents to decondense internally (Ca2+-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small
extent, but with increasing frequency as [Ca2+]
i
signals were reduced by [Ca2+]
o
chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components
(without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca2+]
i
signal was generated by caffeine stimulation (with Ca2+
i
and Ca2+
o
available). We conclude the following. (i) Caffeine can mobilize Ca2+ from cortical stores independent of the presence of Ca2+
o
. (ii) To yield adequate signals for normal exocytosis, Ca2+ release and Ca2+ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca2+]
i
increase entails caffeine-mediated access of Ca2+ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation
in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply
different threshold [Ca2+]
i
-values for membrane fusion and contents discharge.
Received: 23 May 1997/Revised: 18 August 1997 相似文献
18.
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
19.
Ross WN Nakamura T Watanabe S Larkum M Lasser-Ross N 《Cellular and molecular neurobiology》2005,25(2):283-295
Synaptically activated postsynaptic [Ca2+]i increases occur through three main pathways: Ca2+ entry through voltage-gated Ca2+ channels, Ca2+ entry through ligand-gated channels, and Ca2+ release from internal stores. The first two pathways have been studied intensively; release from stores has been the subject of more recent investigations.Ca2+ release from stores in CNS neurons primarily occurs as a result of IP3 mobilized by activation of metabotropic glutamatergic and/or cholingergic receptors coupled to PLC. Ca2+ release is localized near spines in Purkinje cells and occurs as a wave in the primary apical dendrites of pyramidal cells in the hippocampus and cortex. The amplitude of the [Ca2+]i increase can reach several micromolar, significantly larger than the increase due to backpropagating spikes.The large amplitude, long duration, and unique location of the [Ca2+]i increases due to Ca2+ release from stores suggests that these increases can affect specific downstream signaling mechanisms in neurons. 相似文献
20.
Changes in the local environment such as pH (acidosis/alkalosis), temperature (hypothermia/hyperthermia), and agonist (glutamate)
can adversely affect neuronal function, and are important factors in clinical situations such as anesthesia and intensive
care. Regulation of intracellular Ca2+ ([Ca2+]i) is key to neuronal function. Stromal interaction molecule (STIM1) has been recently recognized to trigger store-operated
Ca2+ entry (SOCE), an important component of [Ca2+]i regulation. Using differentiated, fura-2 loaded rat pheochromocytoma (PC12) cells transfected with small interference RNA
for STIM1 (or vehicle), we examined the role of STIM1 in SOCE sensitivity to temperature, pH, and glutamate. SOCE was triggered
following endoplasmic reticulum depletion. Cells were washed and exposed to altered pH (6.0–8.0), altered temperature (34–40°C),
or to glutamate. In non-transfected cells, SOCE was inhibited by acidosis or hypothermia, but increased with alkalosis and
hyperthermia. Increasing glutamate concentrations progressively stimulated SOCE. STIM1 siRNA decreased SOCE at normal temperature
and pH, and substantially decreased sensitivity to acidosis and hypothermia, eliminating the concentration-dependence to glutamate.
Sensitivity of SOCE to these environmental parameters was less altered by decreased extracellular Ca2+ alone (with STIM1 intact). We conclude that STIM1 mediates exquisite susceptibility of SOCE to pH, temperature, and glutamate:
factors that can adversely affect neuronal function under pathological conditions. 相似文献