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1.
Depolarization-evoked increases in intraterminal free Ca2+ are required for the induction of neurotransmitter release from nerve terminals. Although the mechanisms that regulate the voltage-induced accumulation of presynaptic Ca2+ remain obscure, there is evidence that the phospholipase-dependent accumulation of arachidonic acid, or its metabolites, may be involved. Therefore, fura-2 loaded hippocampal mossy fiber nerve endings were used to investigate the relationships between membrane depolarization, lipid metabolism and presynaptic Ca2+ availability. It was observed that depolarization of the nerve terminals with KCl induced an increase in intraterminal free calcium that was inhibited more than 90% by a combination of voltage-sensitive Ca2+ channel blockers. In addition, the K+-dependent effects on Ca2+ concentrations were attenuated in the presence of phospholipase A2 inhibitors, but were mimicked by the phospholipase A2 activator melittin and exogenous arachidonic acid. Both the melittin- and arachidonic acid-induced increases in presynaptic Ca2+ were reduced by voltage-sensitive Ca2+ channel blockers. The stimulatory effects of arachidonic acid appeared to be independent of its further metabolism to prostaglandins. In fact, inhibition of either cyclooxygenase or lipoxygenase pathways resulted in a potentiation of the depolarization-evoked increase in intraterminal free Ca2+. From these results, we propose that some portion of the depolarization-evoked increase in intraterminal free calcium depends on the activation of phospholipase A2 and the subsequent accumulation of unesterified arachidonic acid. 相似文献
2.
Respiring rat liver mitochondria are known to spontaneously release the Ca2+ taken up when they have accumulated Ca2+ over a certain threshold, while Sr2+ and Mn2+ are well tolerated and retained. We have studied the interaction of Sr2+ with Ca2+ release. When Sr2+ was added to respiring mitochondria simultaneously with or soon after the addition of Ca2+, the release was potently inhibited or reversed. On the other hand, when Sr2+ was added before Ca2+, the release was stimulated. Ca2+-induced mitochondrial damage and release of accumulated Ca2+ is generally believed to be due to activation of mitochondrial phospholipase A (EC 3.1.1.4.) by Ca2+. However, isolated mitochondrial phospholipase A activity was little if at all inhibited by Sr2+. The Ca2+ -release may thus be triggered by some Ca2+ -dependent function other than phospholipase. 相似文献
3.
An enhancement of glutamate release from hippocampal neurons has been implicated in long-term potentiation, which is thought to be a cellular correlate of learning and memory. This phenomenom appears to be involved the activation of protein kinase C and lipid second messengers have been implicated in this process. The purpose of this study was to examine how lipid-derived second messengers, which are known to potentiate glutamate release, influence the accumulation of intraterminal free Ca2+, since exocytosis requires Ca2+ and a potentiation of Ca2+ accumulation may provide a molecular mechanism for enhancing glutamate release. The activation of protein kinase C with phorbol esters potentiates the depolarization-evoked release of glutamate from mossy fiber and other hippocampal nerve terminals. Here we show that the activation of protein kinase C also enhances evoked presynaptic Ca2+ accumulation and this effect is attenuated by the protein kinase C inhibitor staurosporine. In addition, the protein kinase C-dependent increase in evoked Ca2+ accumulation was reduced by inhibitors of phospholipase A2 and voltage-sensitive Ca2+ channels, as well as by a lipoxygenase product of arachidonic acid metabolism. That some of the effects of protein kinase C activation were mediated through phospholipase A2 was also indicated by the ability of staurosporine to reduce the Ca2+ accumulation induced by arachidonic acid or the phospholipase A2 activator melittin. Similarly, the synergistic facilitation of evoked Ca2+ accumulation induced by a combination of arachidonic acid and diacylglycerol analogs was attenuated by staurosporine. We suggest, therefore, that the protein kinase C-dependent potentiation of evoked glutamate release is reflected by increases in presynaptic Ca2+ and that the lipid second messengers play a central role in this enhancement of chemical transmission processes. 相似文献
4.
Zhang Yu-an Takashi Imanishi Tetsuyuki Wada Seiji Ichida 《Neurochemical research》1999,24(8):1059-1066
The proportions of calcium (Ca2+) channel subtypes in chick or rat P2 fraction and NG 108-15 cells were investigated using selective L-, N-, P- and P/Q- type Ca2+ channel blockers. KCl-stimulated 45Ca2+ uptake by chick P2 fraction was blocked by 40~50% using N-type Ca2+ channel blockers [-conotoxin GVIA, aminoglycoside antibiotics and dynorphin A(1–13)], but was not inhibited by P- or P/Q-type blockers (-agatoxin IVA or -conotoxin MVIIC). On the other hand, KCl-stimulated 45Ca2+ uptake by rat P2 fraction was blocked by 30~40% using P- or P/Q-type Ca2+ channel blockers, but was not inhibited by N-type Ca2+ channel blockers. The L-type Ca2+ channel blockers 1,4-dihydropyridines, diltiazem and verapamil, but not calciseptine (CaS), inhibited both KCl-stimulated 45Ca2+ uptake and veratridine-induced 22Na+ uptake by chick or rat P2 fraction with similar IC50 values. CaS did not have any effect on 45Ca2+ uptake by either chick or rat P2 fraction. In NG108-15 cells, CaS, -agatoxin IVA and -conotoxin MVIIC, but not -conotoxin GVIA, inhibited KCl-stimulated 45Ca2+ uptake by 30–40%. Various combinations of these Ca2+ channel blockers had no significant additional effects in chick or rat P2 fraction or NG 108-15 cells. These findings suggest that KCl-stimulated 45Ca2+ uptake by chick or rat P2 fraction and NG 108-15 cells is a convenient and useful model for screening whether or not natural or synthetic substances have selective effects as L-, N-, P-, or P/Q- type Ca2+ channel antagonists or agonists. 相似文献
5.
O. DeIbono 《The Journal of membrane biology》1995,146(1):91-99
Ca2+ transients and the rate of Ca2+ release (dCaREL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca2+ indicators. Single pulse experiments with different returning potentials showed that Ca2+ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca2+ removal (dCaREM/dt) was studied by fitting the decaying phase of the Ca2+ transient (Melzer, Ríos & Schneider, 1986) and dCaREL/dt was calculated as the difference between dCa/dt and dCaREM/dt. The fast Ca2+ release decayed as a consequence of Ca2+ inactivation of Ca2+ release. Double pulse experiments showed inactivation of the fast Ca2+ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca2+ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca2+]i and the inhibited amount of Ca2+ release was 0.98. The [Ca2+]i for 50% inactivation of dCaREL/dt was 0.25 m, and the minimum number of sites occupied by Ca2+ to inactivate the Ca2+ release channel was 3.0. These data support Ca2+ binding and inactivation of SR Ca2+ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine. 相似文献
6.
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
7.
Shmuel Muallem Timothy Beeker Stephen J. Pandol 《The Journal of membrane biology》1988,102(2):153-162
Summary The relative contributions of the Na+/Ca2+ exchange and the plasma membrane Ca2+ pump to active Ca2+ efflux from stimulated rat pancreatic acini were studied. Na+ gradients across the plasma membrane were manipulated by loading the cells with Na+ or suspending the cells in Na+-free media. The rates of Ca2+ efflux were estimated from measurements of [Ca2+]
i
using the Ca2+-sensitive fluorescent dye Fura 2 and45Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca2+ efflux is described by a single exponential function under control, Na+-loaded, and Na+-depleted conditions. Manipulation of Na+ gradients had no effect on the hormone-induced increase in [Ca2+]
i
. The results indicate that Ca2+ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca2+ pump. The effects of several cations, which were used to substitute for Na+, on cellular activity were also studied. Choline+ and tetramethylammonium+ (TMA+) released Ca2+ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na+, TMA+, choline+, or N-methyl-d-glucamine+ (NMG+) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG+, choline+, and TMA+ inhibited amylase release by 55, 40 and 14%, respectively. NMG+ also increased the Ca2+ permeability of the plasma membrane. Thus, to study Na+ dependency of cellular function, TMA+ is the preferred cation to substitute for Na+. The stimulatory effect of TMA+ can be blocked by atropine. 相似文献
8.
Angelo O. Rosa Stanley I. Rapoport 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2009,1791(8):697-705
Docosahexaenoic acid (DHA) and arachidonic acid (AA) are found in high concentrations in brain cell membranes and are important for brain function and structure. Studies suggest that AA and DHA are hydrolyzed selectively from the sn-2 position of synaptic membrane phospholipids by Ca2+-dependent cytosolic phospholipase A2 (cPLA2) and Ca2+-independent phospholipase A2 (iPLA2), respectively, resulting in increased levels of the unesterified fatty acids and lysophospholipids. Cell studies also suggest that AA and DHA release depend on increased concentrations of Ca2+, even though iPLA2 has been thought to be Ca2+-independent. The source of Ca2+ for activation of cPLA2 is largely extracellular, whereas Ca2+ released from the endoplasmic reticulum can activate iPLA2 by a number of mechanisms. This review focuses on the role of Ca2+ in modulating cPLA2 and iPLA2 activities in different conditions. Furthermore, a model is suggested in which neurotransmitters regulate the activity of these enzymes and thus the balanced and localized release of AA and DHA from phospholipid in the brain, depending on the primary source of the Ca2+ signal. 相似文献
9.
Importance of Ca2+ influx by Na+/Ca2+ exchange under normal and sodium-loaded conditions in mammalian ventricles 总被引:2,自引:0,他引:2
Satoh H Mukai M Urushida T Katoh H Terada H Hayashi H 《Molecular and cellular biochemistry》2003,242(1-2):11-17
Na+/Ca2+ exchange (NCX) is a major Ca2+ extrusion system in cardiac myocytes, but can also mediate Ca2+ influx and trigger sarcoplasmic reticulum Ca2+ release. Under conditions such as digitalis toxicity or ischemia/reperfusion, increased [Na+]i may lead to a rise in [Ca2+]i through NCX, causing Ca2+ overload and triggered arrhythmias. Here we used an agent which selectively blocks Ca2+ influx by NCX, KB-R7943 (KBR), and assessed twitch contractions and Ca2+ transients in rat and guinea pig ventricular myocytes loaded with indo-1. KBR (5 M) did not alter control steady-state twitch contractions or Ca2+ transients at 0.5 Hz in rat, but significantly decreased them in guinea pig myocytes. When cells were Na+-loaded by perfusion of strophanthidin (50 M), the addition of KBR reduced diastolic [Ca2+]i and abolished spontaneous Ca2+ oscillations. In guinea pig papillary muscles exposed to substrate-free hypoxic medium for 60 min, KBR (10 M applied 10 min before and during reoxygenation) reduced both the incidence and duration of reoxygenation-induced arrhythmias. KBR also enhanced the recovery of developed tension after reoxygenation. It is concluded that (1) the importance of Ca2+ influx via NCX for normal excitation-contraction coupling is species-dependent, and (2) Ca2+ influx via NCX may be critical in causing myocardial Ca2+ overload and triggered activities induced by cardiac glycoside or reoxygenation. 相似文献
10.
The steady-state levels of Ca2+ within the endoplasmic reticulum (ER) and the transport of 45Ca2+ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca2+-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca2+ level in the ER was measured using the Ca2+-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca2+ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of 45Ca2+ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA3) and Ca2+. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of 45Ca2+ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that 45Ca2+ transport was into the ER. The sensitivity of 45Ca2+ transport to inhibitors and the Km of 45Ca2+ uptake for ATP and Ca2+ transport in the microsomal fraction of barley aleurone cells. The rate of 45Ca2+ transport is stimulated several-fold by treatment with GA3. This effect of GA3 is mediated principally by an effect on the activity of the Ca2+ transporter rather than on the amount of ER.Abbreviations CCR
cytochrome-c reductase
- DCCD
dicyclohexylcarbodiimide
- EGTA
ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid
- ER
endoplasmic reticulum
- FCCP
carbonylcyanide p-trifluoromethoxyphenyl hydrazone
- GA3
gibberellic acid
- IDPase
inosine diphosphatase
- Mon
monensin 相似文献
11.
Sergio de la Fuente 《BBA》2010,1797(10):1727-1735
We have investigated the kinetics of mitochondrial Ca2+ influx and efflux and their dependence on cytosolic [Ca2+] and [Na+] using low-Ca2+-affinity aequorin. The rate of Ca2+ release from mitochondria increased linearly with mitochondrial [Ca2+] ([Ca2+]M). Na+-dependent Ca2+ release was predominant al low [Ca2+]M but saturated at [Ca2+]M around 400 μM, while Na+-independent Ca2+ release was very slow at [Ca2+]M below 200 μM, and then increased at higher [Ca2+]M, perhaps through the opening of a new pathway. Half-maximal activation of Na+-dependent Ca2+ release occurred at 5-10 mM [Na+], within the physiological range of cytosolic [Na+]. Ca2+ entry rates were comparable in size to Ca2+ exit rates at cytosolic [Ca2+] ([Ca2+]c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca2+]c. As a consequence, the presence of [Na+] considerably reduced the rate of [Ca2+]M increase at [Ca2+]c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca2+]c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca2+]M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca2+ buffering, and comparison of our results with data on total mitochondrial Ca2+ fluxes indicate that the mitochondrial Ca2+ bound/Ca2+ free ratio is around 10- to 100-fold for most of the observed [Ca2+]M range and suggest that massive phosphate precipitation can only occur when [Ca2+]M reaches the millimolar range. 相似文献
12.
Summary This communication reports the kinetics of the Na+/ Ca2+ exchanger and of the plasma membrane (PM) Ca2+ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca2+ extrusion and its Na+ dependence for concentrations of cytoplasmic free Ca2+ ([Ca2+]cyt) in the 1–10-m range. The PM Ca2+ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol.
104:147–163) for [Ca2+]cyt] 1.5 m with the fluorescent Ca2+ indicator quin2 (K
d= 115 nm). That study determined that the PM Ca2+ pump in the basal state has a V
max = 0.098 mm/min, a K
m= 80 nm and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca2+]cyt with the intracellular Ca2+ probe, rhod2 (K
d= 500 nm), which has almost a fivefold lower affinity for Ca2+. An Appendix also describes the Mg2+ and pH dependence of the K
dand fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca2+ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca2+ extrusion into a Ca2+-free medium (above citation) or (ii) by the newly developed ionomycin short-circuit method, which determines the ionomycin concentration necessary to short circuit the PM Ca2+ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca2+ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca2+ with a K
m= 0.97 ± 0.31 m and Vmax = 1.0 ± 0.6 mm/ min. (ii) At high [Ca2+]cyt, the exchanger works at a rate 10 times as large as the basal V
max of the PM Ca2+ extrusion pump. (iii) The exchanger can work in reverse after Na+ loading of the cytoplasm by monensin. (iv) The PM Ca2+ extrusion pump is activated by exposure to [Ca2+]cyt 1.5 m for 20–50 sec. Activation raises the pump V
max to 1.6 ± 0.6 mm/min and the K
mto 0.55 ± 0.24 m. (v) The Ca2+ buffering capacity of the cytoplasm is 3.6 mm in the 0.1 to 3 m range of [Ca2+]cyt. In summary, the results show that the human platelet can extrude Ca2+ very rapidly at high [Ca2+]cyt. Both the Na+/Ca2+ exchanger and Ca2+ pump activation may prevent inappropriate platelet activation by marginal stimuli.Abbreviations cAMP
cyclic adenosine 3,5-monophosphate
- cGMP
cyclic guanosine 3,5,-monophosphate
- Ca-CAM
calcium calmodulin;
- DT
dense tubules
- B
intrinsic cytoplasmic Ca2+ binding sites
- R
rhod2 or 5-(3,6-bis(dimethylamino)xanth-9-yl)-1-(2-amino-4-hy droxy lphenoxy)-2-(2-amino-5-methylphen- oxy)ethane-N,N,NN-tetraacetic acid
- [Ca2+]cyt
cytoplasmic Ca2+ activity
- quin2
2-[[2-bis[(carboxymethyl)amino]-5-methyl-phenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline
- V or Vextrusion
true rate of Ca2+ extrusion
- fura-2
1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,NN-tetraacetic acid
- AM
acetoxymethyl ester
- DMSO
dimethylsulfoxide
- CTC
chlortetracycline
- EGTA
ethyleneglycol-bis(-aminoethyl ether) N,N,N,N- tetraacetic acid
- HEPES
4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid
- NMDG
N-methyl-d-glucamine
- PIPES
1,4-piperazine-bis-(ethanesulfonic acid)
- HPLC
high performance liquid chromatography
- I
fraction of high-affinity rhod2 complexed with Ca2+
-
F
the observed fluorescence
- Fmin
the minimal fluorescence observed in the absence of Ca2+
- Fmax
the maximal fluorescence observed when the dye is saturated with Ca2+
- X1
the fraction of high-affinity dye
-
K
d,1
dissociation constant of high-affinity dye
-
K
d,2
dissociation constant of the low-affinity dye
- -d1/dt
rate of Ca2+ removal from the rhod2-Ca complex;
- -dF/dt
the slope representing the absolute rate of fluorescence decrease in a progress curve
- Fmax
(Fmax — Fmin)cyt difference between maximal and minimal fluorescence for cytoplasmic high affinity form of rhod2
- F50
fluorescence of the high-affinity form ofrhod2for[Ca2+]cyt=50 nM
- [Ca2+]0
external Ca2+concentration
-
K
p
proportionality constant between the total number of Ca2+ ions moved and the change in high-affinity rhod2 complexation to Ca2
- (d[Ca2+]cyt, T)/dt
rate of Ca2+ influx obtained with maximal levels of ionomycin
- kleak
rate constant for passive inward Ca2+ leakage
- kinno
rate constant for ionomycin-mediated Ca2+ influx
- T
total
- [rhod2]cyt,T
total intracellular rhod2 concentration
- [quin2]cyt,T
total intracellular quin2 concentration
- [B]T
total cytoplasmic buffering capacity
- A[Ca2+]cyt,T
total number of Ca2+ ions moved into the cytoplasm
- [rhod2-Ca]cyt, T
change in concentration of total intracellular high-affinity rhod2 complexed to Ca2+
- [B-Ca]T
change in concentration of total cytoplasmic binding sites complexed to Ca2+
- [quin2]cyt, T
change in concentration of total intracellular quinl complexed to Ca2+
-
change in the degree of intracellular quin2 saturation
- 1
change in degree of saturation of cytoplasmic high-affinity rhod2
- 1-/t
rate of change in degree of saturation of cytoplasmic high affinityrhod2
- Vobs
observed rate of Ca2+ removal from the rhod2-Ca complex
- V8.3 m
the rate of Ca2+ removal from the high affinity rhod2-Ca complex at [Ca2+]cyt = 8.3 m
- /t
rate of change in of the degree of quin2 saturation
- [Ca2+]cytT/t
initial linear rate of ionomycin-mediated Ca2+ influx
- EC50
effective concentration giving a half-maximal effect
- [Na+]cyt
cytoplasmic Na+ activity
- CAM
calmodulin
- ACN
acetonitrile
- TFA
trifuloroacetic acid 相似文献
13.
Sugita Kenji Mörk Ann-Christin Zhang Guo H. Martinez J. Ricardo 《Molecular and cellular biochemistry》1999,198(1-2):39-46
The expression of protein kinase C (PKC) isoforms and the modulation of Ca2+ mobilization by PKC were investigated in the human submandibular duct cell line A253. Three new PKC (nPKC) isoforms (, , and ) and one atypical PKC (aPKC) isoform () are expressed in this cell line. No classical PKC (cPKC) isoforms were present. The effects of the PKC activator phorbol 12-myristate-13-acetate (PMA) and of the PKC inhibitors calphostin C (CC) and bisindolymaleimide I (BSM) on inositol 1,4,5-trisphosphate (IP3) and Ca2+ responses to ATP and to thapsigargin (TG) were investigated. Pre-exposure to PMA inhibited IP3 formation, Ca2+ release and Ca2+ influx in response to ATP. Pre-exposure to CC or BSM slightly enhanced IP3 formation but inhibited the Ca2+ release and the Ca2+ influx induced by ATP. In contrast, pre-exposure to PMA did not modify the Ca2+ release induced by TG, but reduced the influx of Ca2+ seen in the presence of this Ca2+-ATPase inhibitor. These results suggest that PKC modulates elements of the IP3/Ca2+ signal transduction pathway in A253 cells by (1) inhibiting phosphatidylinositol turnover and altering the sensitivity of the Ca2+ channels to IP3, (2) altering the activity, the sensitivity to inhibitors, or the distribution of the TG-sensitive Ca2+ ATPase, and (3) modulating Ca2+ entry pathways. 相似文献
14.
Naranjan S. Dhalla Vincenzo Panagia Naoki Makino Robert E. Beamish 《Molecular and cellular biochemistry》1988,82(1-2):75-79
In order to identify defects in Na+-Ca2+ exchange and Ca2+-pump systems in cardiomyopathic hearts, the activities of sarcolemmal Na+-dependent Ca2+ uptake, Na+-induced Ca2+ release, ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase were examined by employing cardiomyopathic hamsters (UM-X7.1) and catecholamine-induced cardiomyopathy produced by injecting isoproterenol into rats. The rates of Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase activities of sarcolemmal vesicles from genetically-linked cardiomyopathic as well as catecholamine-induced cardiomyopathic hearts were decreased without any changes in Na+-induced Ca2+-release. Similar results were obtained in Ca2+-paradox when isolated rat hearts were perfused for 5 min with a medium containing 1.25 mM Ca2+ following a 5 min perfusion with Ca2+-free medium. Although a 2 min reperfusion of the Ca2+-free perfused hearts depressed sarcolemmal Ca2+-pump activities without any changes in Na+-induced Ca2+-release, Na+-dependent Ca2+ uptake was increased. These results indicate that alterations in the sarcolemmal Ca2+-efflux mechanisms may play an important role in cardiomyopathies associated with the development of intracellular Ca2+ overload. 相似文献
15.
Yang KT Chen WP Chang WL Su MJ Tsai KL 《Biochemical and biophysical research communications》2005,331(2):603-613
Arachidonic acid (AA) plays important physiological or pathophysiological roles. Here, we show in cultured rat astrocytes that: (i) endothelin-1 or thapsigargin (Tg) induces store-depleted activated Ca2+ entry (CCE), which is inhibited by 2-aminoethoxydiphenyl borane (2-APB) or La3+; (ii) AA (10 μM) and other unsaturated fatty acids (8,11,14-eicosatrienoic acid and γ-linoleic acid) have an initial inhibitory effect on the CCE, due to AA- or fatty acid-induced internal acid load; (iii) after full activation of CCE, AA induces a further Ca2+ influx, which is not inhibited by 2-APB or La3+, indicating that AA activates a second Ca2+ entry pathway, which coexists with CCE; and (iv) Tg or AA activates two independent and co-existing non-selective cation channels and the Tg-induced currents are initially inhibited by addition of AA or weak acids. A possible pathophysiological effect of the AA-induced [Ca]i overload is to cause delayed cell death in astrocytes. 相似文献
16.
The effect of regucalcin, a Ca2+-binding protein, on Ca2+ transport system in rat renal cortex microsomes was investigated. The presence of regucalcin (10-8 to 10-6 M) in the reaction mixture caused a significant increase in Ca2+-ATPase activity and ATP-dependent45 Ca2+ uptake in the microsomes. Regucalcin (10-7 M) increased Ca2+-ATPase activity independently of increasing concentrations of CaCl_2. The microsomal Ca2+-ATPase activity and45 Ca2+ uptake were markedly decreased by the presence of vanadate (0.1 mM) or N-ethylmaleimide (NEM; 5 mM) in the absence or presence of regucalcin. Dithiothreitol (DTT; 5 mM) markedly elevated Ca2+-ATPase activity and 45Ca2+ uptake in the microsomes. The DTT effects were not further enhanced by regucalcin (10-7 M). Meanwhile, the microsomal Ca2+-ATPase activity and 45Ca2+ uptake were significantly decreased by the presence of dibutyryl cyclic AMP (DcAMP; 10-5 and 10-3 M) or inositol 1,4, 5-trisphosphate (IP3; 10-7 and 10-5 M). The effect of regucalcin (10-7 M) on Ca2+ ATPase activity and 45Ca2+ uptake was weakened in the presence of DcAMP or IP3. The present results demonstrate that regucalcin has a stimulatory effect on ATP-dependent Ca2+ uptake in the microsomes of rat renal cortex due to acting on the thiol groups of Ca2+-ATPase. 相似文献
17.
Cefaratti C 《Molecular and cellular biochemistry》2007,295(1-2):241-247
Isolated hepatocytes release 2–3 nmol Mg2+/mg protein or ~10% of the total cellular Mg2+ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During
Mg2+ release, a quantitatively similar amount of Ca2+ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg2+:1Ca2+. Calcium induced Mg2+ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg2+:1Ca2+ exchange ratio. The uptake of Ca2+ for the release of Mg2+ occurs in the absence of significant changes in Δψ as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium
(TPP+) electrode or 3H-TPP+. Collapsing the Δψ by high concentrations of TPP+ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca2+-induced Mg2+ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca2+ uptake and Mg2+ release. These data demonstrate the operation of an electroneutral Ca2+/Mg2+ exchanger which represents a novel pathway for Ca2+ accumulation in liver cells following adrenergic receptor stimulation.
This work was supported by National Institutes of Health Grant HL 18708. 相似文献
18.
Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca2+ transport have been examined in intact human red blood cells. Active Ca2+ efflux was determined from the initial rate of45Ca2+ loss after CoCl2 was added to block Ca2+ loading via the ionophore A23187. Ca2+-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca2+ in the presence of A23187. The apparent Ca affinity of active Ca2+ efflux (K
0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca2+-ATPase assay (K
0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca2+ efflux and Ca2+-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg2+-depleted cells, and completely restored by reintroduction of intracellular Mg2+. Active Ca2+ efflux was inhibited almost completely by raising external CaCl2 (but not MgCl2) to 20mm, probably by interaction of Ca2+ at the externally oriented E2P conformation of the pump. Cd2+ was more potent than Ca2+ in this inhibition, while Mn2+ was less potent and 10mm Ba2+ was without effect. A Ca2+: proton exchange mechanism for active Ca2+ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca2+ efflux at least twofold above the efflux rate at pH 7.8 Ca2+ transport was not affected by decreasing the membrane potential across the red cell. 相似文献
19.
M. V. Wright N. Elwess J. van Houten 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1993,163(4):288-296
Intracellular Ca2+ levels in Paramecium must be tightly controlled, yet little is understood about the mechanisms of control. We describe here indirect evidence that a phosphoenzyme intermediate is the calmodulin-regulated plasma membrane Ca2+ pump and that a Ca2+-ATPase activity in pellicles (the complex of cell body surface membranes) is the enzyme correlate of the plasma membrane pump protein. A change in Ca2+ pump activity has been implicated in the chemoresponse of paramecia to some attractant stimuli. Indirect support for this is demonstrated using mutants with different modifications of calmodulin to correlate defects in chemoresponse with altered Ca2+ homeostasis and pump activity.Abbreviations EGTA
ethyleneglycol tetra-acetate
- ER
endoplasmic reticulum
- IBMX
isobutyl methylxanthine
-
I
che
index of chemokinesis
- Mops
3-[N-morpholino] propanesulfonic acid
- PEI
phosphoenzyme intermediate
- STEN
sucrose, TRIS, EDTA, sodium chloride
- TCA
trichloroacetic acid
- TRIS
tris[hydroxymethyl] aminomethane 相似文献
20.
The cytoplasmic Ca2+ concentration ([Ca2+]cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether
capacitative Ca2+ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca2+]cyt. We measured changes of [Ca2+]cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca2+] from 1.3 mM to 5 mM induced an incrase in [Ca2+]cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd3+, 10 μM La3+ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca2+ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca2+) resulted in a decrease of [Ca2+]cyt from 105±10 nM to 88.5±10 nM with La3+, from 103±12 to 89±12 nM with Gd3+ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca2+ entry beside its function in Ca2+ signaling contributes to the regulation of resting [Ca2+]cyt. 相似文献