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1.
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
2.
Summary To control the intracellular free Ca2+ concentration from the cell exterior, pollen tubes ofLilium longiflorum were treated with a Ca2+ ionophore, A23187. Cytoplasmic streaming was inhibited when the free Ca2+ concentration of the external medium ([Ca2+]) was raised to 5×10–6 M or higher. At [Ca2+] below 1×10–6 M, the rhodamine-phalloidin stained actin filaments appeared straight and thin. However, at [Ca2+] which inhibited cytoplasmic streaming, the actin filaments appeared fragmented. In pollen tubes, Ca2+ regulation of cytoplasmic streaming may be linked not only to myosin (Shimmen 1987) but also to actin.Abbreviations ATP
adenosine-5-triphosphoric acid
- [Ca2+]
concentration of free Ca2+
- EGTA
ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- PIPES
piperazine-N,N-bis(2-ethanesulfonic acid)
- Rh-ph
rhodamine-conjugated phalloidin 相似文献
3.
Summary Using aequorin luminescence, we observed a distinct oscillation in Ca2+ levels in the supernatant of the homogenate ofPhysarum plasmodium. Ca2+ oscillation continued for 10–120 minutes, with a period coinciding with that of the contraction rhythm of a plasmodium.Abbreviations EDTA
Ethylenediaminetetraacetic acid
- EGTA
Ethyleneglycol-bis-(-aminoethylether)-N,N-tetraacetic acid
- PIPES
Piperazine-N,N-bis-(2-ethane sulfonic acid)
- DTT
Dithiothreitol
The present work was supported by Grants-in Aid from the Ministry of Education, Science and Culture, Japan. 相似文献
4.
Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca2+. To determine if Ca2+ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca2+, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca2+, was observed only in the perfusion solution containing Ca2+, indicating that myosin is responsible for the inhibitory effect of Ca2+ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca2+ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP
adenosine 5-triphosphate
- DTT
dithiothreitol
- EDTA
ethylenediaminetetraacetic acid
- EGTA
ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid
- PIPES
piperazine-N,N-bis(2-ethanesulfonic acid) 相似文献
5.
Ursula Meindl 《Protoplasma》1982,112(1-2):138-141
Summary During the stage of pore formation developing cells ofMicrasterias denticulata show a patterned distribution of fluorescent dots on the plasma membrane after treatment with chlorotetracycline. The center-to-center spacing of these dots corresponds with the distances between the individual cell wall pores ofMicrasterias. Therefore it is supposed that the patterned distribution of pores and their formation which is mediated by special pore vesicles are related to local accumulations of membrane-associated Ca2+. Membrane-associated Ca2+ seems not only to be functional in tip growth but to be a general mediator for recognition and fusion processes between various vesicles and the plasma membrane. 相似文献
6.
Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line 总被引:1,自引:0,他引:1
Xinjun He Xiaozai Wu R. James Turner Bruce J. Baum 《The Journal of membrane biology》1990,115(2):159-166
Summary We have investigated muscarinic receptor-operated Ca2+ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca2+ release and extracellular Ca2+ entry. The carbachol (Cch) dose response of intracellular Ca2+ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K
0.510 or 30 m, depending on the method for [Ca2+]
i
determination). However, similar data for Ca2+ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca2+ release and a second higher affinity site withK
0.52.5 m. In addition, the Ca2+ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K+ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca2+ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca2+]
i
of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca2+ channel blocker La3+ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca2+ in these cells by increasing Ca2+ entry. Organic Ca2+ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca2+ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K+ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP3) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP3 formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca2+ entry in HSG-PA cells. One is associated with IP3 formation and intracellular Ca2+ release and is independent of membrane potential; the other is less dependent on IP3 formation and intracellular Ca2+ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating. 相似文献
7.
C. Bandeira-Duarte C. A. M. Carvalho E. J. Cragoe Jr A. P. Carvalho 《Neurochemical research》1990,15(3):313-320
Preparations of synaptosomes isolated in sucrose or in Na+-rich media were compared with respect to internal pH (pH1), internal Ca2+ concentration ([Ca2+]i), membrane potential and45Ca2+ uptake due to K+ depolarization and Na+/Ca2+ exchange. We found that synaptosomes isolated in sucrose media have a pHi of 6.77±0.04 and a [Ca2+]i of about 260 nM, whereas synaptosomes isolated in Na+-rich ionic media have a pHi of 6.96±0.07 and a [Ca2+]i of 463 nM, but both types of preparations have similar membrane potentials of about –50 mV when placed in choline media. The sucrose preparation takes up Ca2+ only by voltage sensitive calcium channels (VSCC'S) when K+-depolarized, while the Na+-rich synaptosomes take up45Ca2+ both by VSCC'S and by Na+/Ca2+ exchange. The amiloride derivative 2, 4 dimethylbenzamil (DMB), at 30 M, inhibits both mechanisms of Ca2+ influx, but 5-(N-4-chlorobenzyl)-2, 4 dimethylbenzamil (CBZ-DMB), at 30 M, inhibits the Ca2+ uptake by VSCC'S, but not by Na+/Ca2+ exchange. Thus, DMB and CBZ-DMB permit distinguishing between Ca2+ flux through channels and through Na+/Ca2+ exchange. We point out that the different properties of the two types of synaptosomes studied account for some of the discrepancies in results reported in the literature for studies of Ca2+ fluxes and neurotransmitter release by different types of preparations of synaptosomes.Abbreviations used BCECF
2,7-Biscarboxyethyl-5(6)-carboxyfluorescein
- BCECF/AM
acetoxymethyl ester of BCECF
- [Ca2+]i
Internal free calcium ion concentration
- CBZ-DMB
5-(N-4-chlorobenzyl)-2,4-dimethylbenzamil
- DMB
2, 4-dimethylbenzamil
- DMSO
dimethyl sulfoxide
- Indo-1/AM
acetoxymethyl ester of Indo-1
- MES
2-|N-Morpholino|ethanesulfonic acid
- NMG
N-methyl-D-glucamine
- pHi
internal pH
- TPP+
tetraphenylphosphonium
- p
plasma membrane potential 相似文献
8.
A rapid induction by elicitors of the mRNA encoding CCD-1, a 14 kDa Ca2+-binding protein in wheat cultured cells 总被引:1,自引:0,他引:1
Takezawa D 《Plant molecular biology》2000,42(6):807-817
Intracellular Ca2+ has been implicated in the signal transduction processes during the development of the plant defense system against fungal pathogens. From wheat cultured cells that had been treated with the elicitor derived from Typhula ishikariensis, the ccd-1 gene encoding a 14 kDa Ca2+-binding protein with an acidic amphiphilic feature was isolated. The ccd-1-encoded protein (CCD-1) shares homology to the C-terminal half domain of centrin, a Ca2+-binding protein conserved in eukaryotes. Unlike typical eukaryotic centrins, CCD-1 contains only one Ca2+-binding loop, which corresponds to the one in the fourth EF-hand from the N-terminus of centrin. The recombinant CCD protein expressed in Escherichia coli bound to a phenyl-Sepharose column in the presence of Ca2+ and was eluted out by EGTA. It also showed a Ca2+-dependent electrophoretic mobility shift on the non-denaturing polyacrylamide gel. The ccd-1 mRNA expression was rapidly induced by treatment with fungal and chitosan oligosaccharide elicitors, implying that it might have a role in transducing Ca2+ signals provoked by the elicitors. The expression of the ccd-1 mRNA was induced by treatment with A23187, and the induction was suppressed by La3+ or 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA). This study suggests the involvement of intracellular Ca2+ in the elicitor-induced mRNA expression of a novel class of Ca2+-binding proteins conserved in higher plants. 相似文献
9.
J. P. Arsanto 《Protoplasma》1986,132(3):160-171
Summary In stem ofCicer arietinum, the loss of ribosomes attached to the rough ER cisternae during sieve element ontogeny results in the formation of sieve element reticulum (SER). By enhancing contrast of the SER, the OsFeCN postfixation/staining of material prefixed in glutaraldehyde in presence of calcium enables a good visualization of this membrane system. The pattern of staining in the SER is slightly lower when Mg2+ is substituted for Ca2+. These results support the view that the OsFeCN staining requires divalent cations and that the SER can accumulate Ca2+. The detection of Ca2+ by means of the pyroantimonate method in conjunction with X-ray microanalysis and the cytochemical localization of Ca2+ -ATPase in the SER cisternae provides evidence for Ca2+ sequestration by the SER. On the other hand, Ca2+-binding sites and ATPase activity are localized in P-protein. The ability to bind Ca2+ seems to enable the SER to function as an effective Ca2+ sink which may participate—with the sieve tube plasma membrane and mitochondria—in the maintenance of low Ca2+ concentration in phloem sap. In addition, the close association between P-protein and SER membranes exhibiting Ca2+-binding capabilities suggests that a Ca2+-mediated functional relationship may exist between the two structures. It is postulated that the SER may play a role in putative Ca2+ control of P-protein organization.Abbreviations SER
sieve element reticulum
- ER
endoplasmic reticulum
- P
protein, phloem protein
- OsFeCN
method, osmium tetroxide-ferricyanide method
- EDTA
ethylenediamine tetraacetic acid
- EGTA
ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid
- ATP
adenosine 5-triphosphoric acid
- ATPase
adenosine triphosphatase
- PCMB
p chloromercuribenzoate
- IDP
inosine diphosphate 相似文献
10.
Summary We examined the ionic regulation of tip growth inNeurospora crassa by a combination of electrophysiology and confocal microscopy. To determine if transmembrane ionic fluxes are required for tip growth, we voltage clamped the membrane from –200 to +50 mV. In this voltage range, transmembrane ionic fluxes would either reverse (e.g., K+) or change dramatically (e.g., Ca2+ influx) but had no effect on hyphal growth rates. Therefore, ionic fluxes (including Ca2+ influx) may not be required for tip growth. However, intracellular Ca2+ may still play an obligatory role in tip growth. To assess this possibility, we first increased cytosolic Ca2+ directly by ionophoresis. Elevated Ca2+ induced subapical branch initiation, often multiple tips. At hyphal tips, fluorescence ratio imaging using fluo-3 and fura-red revealed a pronounced tip-high Ca2+ gradient within 10 m of the tip in growing hyphae which was not observed in nongrowing hyphae. Injection of the Ca2+ chelator 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate consistently inhibited growth concomitantly with a depletion of intracellular Ca2+ and dissipation of the tip-high gradient. We conclude that Ca2+ plays a regulatory role in tip initiation and the maintenance of tip growth. Because plasma membrane ionic fluxes do not play a role in tip growth, we suggest that the tip-high Ca2+ gradient is generated from intracellular Ca2+ stores in the ascomyceteN. crassa.Abbreviations BAPTA
1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate
- [Ca2+]i
intracellular Ca2+ concentration
- fluo-3
2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl-4-methyl-2,2-(ethylenedioxy)dianiline-N,N,N,N-tetraacetic acid 相似文献
11.
We showed that 5 M acetylcholine (ACh) and 100 M norepinephrine (NE) cause increases in the total Ca2+ content in acinar cells by 30 and 87% and in the exocytosis intensity by 15 and 20%, respectively. Application of 5 M ACh and 100 M NE increased the free cytosolic Ca2+ concentration ([Ca2+]
i
) by 87 ± 2 and 140 ± 7 nM, respectively. Application of ACh and NE in a Ca2+-free external solution caused a [Ca2+]
i
increase that was 40 and 67% lower than in physiological solution. We postulate that the exocytosis developing upon neural stimulation of the gland results from generation of Ca2+ transients that are spreading from the basal to the apical region of the exocrine cell, where secretory granules are concentrated. 相似文献
12.
Summary Striated Ciliary Roots (SCRs), about 3 m long, are attached to the basal bodies of branchial crown ciliated epithelial cells ofOwenia. These SCRs appear to consist of 5–7-nm diameter filaments organized in a cross-striation pattern with an apparent variable periodicity of 50 to 80 nm. The most exciting observation emerging from this study is the constant and conspicuous close spatial relationship between SCRs and fairly well developed Golgi apparatus. By enhancing contrast and preservation of cell components, the OsFeCN postfixation-staining of material prefixed in glutaraldehyde in the presence of calcium has revealed some fine-structural details within the SCR-Golgi Association. By means of the calcium precipitation method, with antimonate or oxalate in conjunction with X-ray microanalysis, we have identified calcium within SCR dark bands and SCR-associated Golgi bodies. The ability to bind calcium makes the Golgi apparatus a likely candidate for Ca2+ regulation of putative contraction of the SCRs and/or ciliary motility. The slight period variability measured in the SCRs and cytochemical localization of Mg2+, Ca2+-dependent ATPase activities associated with cross striations support the view that theOwenia SCRs may be contractile organelles.The striking and specific close structural association between the Golgi apparatus and the SCR showing Ca2+-binding capabilities suggests that some sort of Ca2+-mediated functional relationship between these organelles may exist.Abbreviations SCR
striated ciliary root
- OsFeCN method
osmium tetroxide-ferricyanide method
- EDTA
ethylenediamine tetraacetic acid
- EGTA
ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid
- ATP
adenosine 5-triphosphoric acid
- ATPase
adenosine triphosphatase
- ASW
artificial sea-water 相似文献
13.
Selectivity of ATP-activated GTP-dependent Ca2+-permeable channels in rat macrophage plasma membrane
A. P. Naumov E. V. Kaznacheyeva Y. A. Kuryshev G. N. Mozhayeva 《The Journal of membrane biology》1995,148(1):91-98
Outside-out configuration of the patch clamp technique was used to test whether an intracellular application of G protein activator (GTPS) affects ATP-activated Ca2+-permeable channels in rat macrophages without any agonist in the bath solution. With 145 mm K+ (pCa 8.0) in the pipette solution, activity of channels permeable to a variety of divalent cations and Na+ was observed and general channel characteristics were found to be identical to those of ATP-activated ones. Absence of extracellular ATP makes it possible to avoid the influence of ATP receptor desensitization and to study the channel selectivity using a number of divalent cations (105 mm) and Na+ (145 mm) as the charge carriers. Permeability sequence estimated by extrapolated reversal potential measurements was: Ca2+ Ba2+ Mn2+ Sr2+ Na+ K+ = 68 30 26 10 3.5 1. Slope conductances (in pS) for permeant ions rank as follows: Ca2+ Sr2+ Na+ Mn2+ Ba2+ = 19 18 14 12 10. Unitary Ca2+ currents display a tendency to saturate with the Ca2+ concentration increase with apparent dissociation constant (K
d
) of 10 mm. No block of Na+ permeation by extracellular Ca2+ in millimolar range was found. The data obtained suggest that (i) activation of some G protein is sufficient to gate the channels without the ATP receptor being occupied, (ii) the ATP receptor activation results in the gating of a special channel with the properties that differ markedly from those of the receptoroperated or voltage-gated Ca2+-permeable channels on the other cell types.DeceasedThe authors are grateful to K. Kiselyov and A. Mamin for technical assistance. The work was supported by the Russian Basic Research Foundation, Grant N 93-04-21722 and was made possible in part by Grant N R4A000 from the International Science Foundation. 相似文献
14.
P. Ballerini R. Ciccarelli P. Di Iorio P. Giuliani F. Caciagli 《Neurochemical research》1995,20(6):697-704
[3H]Purine release from rat striatum astrocyte cultures was studied at 14 days in vitro (DIV). Superfusion of cultures with a Ca2+-free medium +0.5 mM ethylene glycol-bis(-aminoethylether)N,N,N,N-tetracetic acid (EGTA) reduced the electrically evoked [3H]purine release. Nimodipine only at the concentration of 10 M modified [3H]purine outflow whereas 0.1 M -conotoxin and 0.03–0.1 M nitrendipine reduced the evoked one. Superfusion of cultures with 0.1 M -conotoxin +0.1 M nitrendipine antagonized the evoked [3H]purine release similarly to each drug given alone. Neither nitrendipine nor -conotoxin influenced the uptake of45Ca2+ by the cultures. The treatment of cells with the Ca2+ agonist Bay K 8644 did not affect [3H]purine release or the45Ca2+ uptake. The drug did not either alter [Ca2+]i, evaluated by loading the cells with 3 M Fura-2/AM. 10–30 M 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular Ca2+ discharge, significantly reduced the evoked [3H]purine release. On the other hand, 2 M thapsigargin, an inhibitor of the ion store Ca2+ ATPase, was able to increase either the culture [3H]purine release or the [Ca2+]i. Together, the findings indicate that voltage-sensitive calcium channels (VSCCs) of the neuronal N and L-types are not involved in the modulation of [3H]purine release from rat cultured astrocytes whereas Ca2+ coming from intracytoplasmic stores seems to play a prevailing role. Moreover, agents which block VSCCs seem to be able to affect [3H]purine outflow with mechanisms other than VSCC gating. 相似文献
15.
This review will focus on the recent advance in the study of effect of transmembrane Ca2+ gradient on the function of membrane proteins. It consits of two parts: 1. Transmembrane Ca2+ gradient and sarcoplasmic reticulum Ca2+-ATPase; 2. Effect of transmembrane Ca2+ gradient on the components and coupling of cAMP signal transduction pathway. The results obtained indicate that a proper transmembrane Ca2+ gradient may play an important role in modulating the conformation and activity of SR Ca2+-ATPase and the function of membrane proteins involved in the cAMP signal transduction by mediating the physical state change of the membrane phospholipids.Abbreviations Cai
Ca2+ inside vesicles
- Ca0
Ca2+ outside vesicles
- SR
sarcoplasmic reticulum
- PC
phosphatidylcholine
- PS
phosphatidylserine
- PG
phosphatidylglycerol
- PE
phosphatidylethanolamine
- DPH
1,6-diphenyl-1,3,5-hexatriene
- n-AS
n-(9-anthroyloxy) fatty acids
- TMA-DPH
1-(4-trimethylammoniumphenyl)-6)-phenyl-1,3,5-hexatriene
- FCCP
carbonylcyanide-p-trifluoromethoxyphenylhydrazone
- -AR
-adrenergic receptors
- DHA
dihydroalprenolol
- AC
adenylate cyclase
- AC·Lca+–
higher Ca2+ inside vesicles
- AC·Lca– –
lower Ca2+ on both side of vesicles
- AC·Lca++
higher Ca2+ on both side of vesicles
- AC·Lca– +
higher Ca2+ outside vesicles
- cAMP
cyclic adenosine monophosphate
- Gs
stimulatory GTP-binding protein
- GTP
guanosine triposphate
- GTPS
guanosine 50-(3-thiotriphosphate) 相似文献
16.
Rajesh Mahey Michael A. Bridges Sidney Katz 《Molecular and cellular biochemistry》1991,105(2):137-147
Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca2+-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca2+-transporter showed high affinity for Ca2+ (KCa
2+ = 0.04 ± 0.01 M), an apparent requirement for Mg2+ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca2+ or Mg2+ but showed a low affinity for these cations. At low concentrations, Mg2+ appeared to inhibit the Ca2+-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg2+ concentration (0.5–1 mM), a high affinity Ca2+-dependent ATPase activity was observed (KCa
2+ = 0.08 ± 0.02 M). The hydrolytic activity showed little specificity towards ATP. Neither the Ca2+-transport nor high affinity Ca2+-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca2+ (or Mg+)-ATPase activity, the presence of both a high affinity Ca2+-pump and high affinity Ca2+-dependent ATPase. However, the high affinity Ca2+-ATPase activity does not appear to be the biochemical expression of the Ca2+-pump.Abbreviations Ca2+-ATPase
calcium-activated, magnesium-dependent adenosine triphosphatase
- CaM
calmodulin
- CDTA
trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetate
- EDTA
ethylene-diaminetetraacetate
- EGTA
ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetate
- NADPH
reduced form of nicotinamide adenine dinucleotide phosphate 相似文献
17.
Alicia Mimbrera Luis Rivas Faustino Mollinedo Emilio Muñoz Vicente Larraga 《Molecular and cellular biochemistry》1983,56(1):73-80
Summary The combined use of proteolytic digestion and lactoperoxidase catalyzed labelling with [125I] applied to membrane-bound or soluble pure F1-ATPase from Micrococcus lysodeikticus has allowed us to establish the topography of its , , and subunits within the protein molecule and with respect to the plane of the membrane.The subunit is most externally located to the membrane bilayer looking towards the cytoplasmic face, a position consistent with its proposed catalytic role. The and subunits lie in an intermediate layer between the subunits and the membrane, in which the subunit occupies a central position within the F1-ATPase molecule in contact with the subunit. The subunit appears to be tightly bound to the F0 component of the ATPase complex, probably buried in the membrane bilayer. A molecular arrangement of M. lysodeikticus ATPase is proposed that, taking into account the subunit stoichiometry 3 3 2 2 (MW 420 000), accommodates the role assigned to each subunit and most, if not all, the known properties of this bacterial energy-transducing protein. 相似文献
18.
In skeletal and cardiac muscle, contraction is initiated by the rapid release of Ca2+ ions from the intracellular membrane system, sarcoplasmic reticulum. Rapid-mixing vesicle ion flux and planar lipid bilayer-single-channel measurements have shown that Ca2+ release is mediated by a high-conductance, ligand-gated Ca2+ channel. Using the Ca2+ release-specific probe ryanodine, a 30 S protein complex composed of four polypeptides ofM
r 400,000 has been isolated. Reconstitution of the purified skeletal and cardiac muscle 30 S complexes into planar lipid bilayers induced single Ca2+ channel currents with conductance and gating kinetics similar to those of native Ca2+ release channels. Electron microscopy revealed structural similarity with the protein bridges (feet) that span the transverse-tubule-sarcoplasmic reticulum junction. These results suggest that striated muscle contains an intracellular Ca2+ release channel that is identical with the ryanodine receptor and the transverse-tubule-sarcoplasmic reticulum spanning feet structures. 相似文献
19.
Izuo Tsutsui Taka-aki Ohkawa Reiko Nagai Uichiro Kishimoto 《The Journal of membrane biology》1987,96(1):75-84
Summary The steady N shapeI/V curves were obtained by applying slow ramp hyper- and depolarization pulses toChara cells under the voltage-clamp condition. Application of calcium channel blocker, 20 m La3+, to theChara membrane caused, in about 30 min, a marked reduction of the transient inward current and later almost complete blocking of the pump current, while the steady outward current remained almost unaffected. Removal of external Ca2+ with 0.5mm EGTA caused similar results. Application of calmodulin antagonists, 10 m TFP or 20 m W-7, also gave very similar results, i.e., the decrease of the transient inward current and of H+-pump activity. These results suggest that not only the excitatory mechanisms but also the H+-pump activity ofChara membrane are regulated by calmodulin within a comparatively narrow range of internal Ca2+ level. 相似文献
20.
Summary A systematic study was made of the action of 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) on active Ca2+ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca2+-selective electrodes or use of tracer45Ca2+. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS. Ca2+ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K+ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca2+ pump, exchanging one Ca2+ ion for one proton. 相似文献