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1.
Summary The ATP-dependent Ca2+ transport activity (T. Takuma, B.L. Kuyatt and B.J. Baum,Biochem. J.
227:239–245, 1985) exhibited by inverted basolateral membrane vesicles isolated from rat parotid gland was further characterized. The activity was dependent on Mg2+. Phosphate (5mm), but not oxalate (5mm), increased maximum Ca2+ accumulation by 50%. Half-maximal Ca2+ transport was achieved at 70nm Ca2+ in EGTA-buffered medium while maximal activity required >1 m Ca2+ (V
max=54 nmol/mg protein/min). Optimal rates of Ca2+ transport were obtained in the presence of KCl, while in a KCl-free medium (mannitol or sucrose) 40% of the total activity was achieved, which could not be stimulated by FCCP. The initial rate of Ca2+ transport could be significantly altered by preimposed membrane potentials generated by K+ gradients in the presence of valinomycin. Compared to the transport rate in the absence of membrane potential, a negative (interior) potential stimulated uptake by 30%, while a positive (interior) potential inhibited uptake. Initial rates of Ca2+ uptake could also be altered by imposing pH gradients, in the absence of KCl. When compared to the initial rate of Ca2+ transport in the absence of a pH gradient, pH
i
=7.5/pH
o
=7.5; the activity was 60% higher in the presence of an outwardly directed pH gradient, pH
i
=7.5/pH
o
=8.5; while it was 80% lower when an inwardly directed pH gradient was imposed, pH
i
=7.5/pH
o
=6.2. The data show that the ATP-dependent Ca2+ transport in BLMV can be modulated by the membrane potential, suggesting therefore that there is a transfer of charge into the vesicle during Ca2+ uptake, which could be compensated by other ion movements. 相似文献
2.
The effects of external pH (pH
out) variations on the Na+ and on the Ca2+ dependent fractions of the evoked amino acid neurotransmitter release were separately investigated, using GABA as a model transmitter. In [3H]GABA loaded mouse brain synaptosomes, the external acidification (pH
out6.0) markedly decreased the Na+ dependent fraction of [3H]GABA release evoked by veratridine (10 M) in the absence of external Ca2+, as well as the Ca2+ dependent fraction of [3H]GABA release evoked by high (20 mM) K+ in the absence of external Na+. The depolarization-induced elevation of [Na
i
] (monitored in synaptosomes loaded with the Na+ indicator dye, SBFI) and the depolarization-induced elevation of [Ca
i
] (monitored in synaptosomes loaded with the Ca2+ indicator dye fura-2) were also markedly decreased at pH
out 6. On the contrary, the external alkalinization (pH
out 8) facilitated all the above responses. A slight increase of the baseline release of the [3H]GABA was observed when pH
out was changed from 7.4 to 8. This effect was only observed in the presence of Ca2+. pH
out changes from 7.4 to 6 or to 7 did not modify the baseline release of the transmitter. All the effects of pH
out variations on [3H]GABA release were independent on the presence of HCO-3. It is concluded that external H+ regulate amino acid neurotransmitter release by their actions on presynaptic Na+ channels, as well as on presynaptic Ca2+ channels. 相似文献
3.
The influence of cytosolic pH (pHi) in controlling K+-channel activity and its interaction with cytosolic-free Ca2+ concentration ([Ca2+]i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K+-channel currents were recorded under voltage clamp while pHi or [Ca2+]i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K+ concentration, current through inward-rectifying K+ channels (IK,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na+-acetate outside imposed acid loads, lowering pHi from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pHi was independent of the weak acid used, and indicated a H+-buffering capacity which rose from 90 mM H+/pH unit near 7.5 to 160 mM H+/pH unit near pHi 7.0. With acid-going pHi, (IK,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pHi lasting 3–5 min and was accompanied by a reduction in (IK,in) before recovery of the initial resting pHi and current amplitude. The pHi-sensitivity of the current was consistent with a single, titratable site for H+ binding with a pKa near 6.3. Acid pHi loads also affected current through the outward-rectifying K+ channels (IK,out) in a manner antiparallel to (IK,in) The effect on IK, out was also scalar, but showed an apparent pKa of 7.4 and was best accommodated by a cooperative binding of two H+. Parallel measurements showed that Na+-butyrate loads were generally without significant effect on [Ca2+]i, except when pHi was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca2+]i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pHi changes and K+-channel responses. The action on [Ca2+]i coincided with a greater variability in (IK,in) stimulation evident at pHi values around 7.0 and below, and with negative displacements in the voltage-dependence of (IK,in) gating. These results distinguish the actions of pHi and [Ca2+]i in modulating (IK,in) they delimit the effect of pHi to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pHi as a second messenger capable of acting in parallel with, but independent of [Ca2+]i in controlling the K+ channels.Abbreviations BCECF
2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein
- [Ca2+]i
cytosolic free Ca2+ concentration
- gK
ensemble (steady-state) K+-channel conductance
- IK,out, IK,in
outward-, inward-rectifying K+ channel (current)
- IN
current-voltage (relation)
- Mes
2-(N-morpholinolethanesulfonic acid
- pHi
cytosolic pH
- V
membrane potential 相似文献
4.
Summary Intracellular pH (pH
i
) and intracellular Ca2+ ([Ca2+]
i
) were determined inChironomus salivary gland cells under various conditions of induced uncoupling. pH
i
was measured with aThomas-type microelectrode, changes in [Ca2+]
i
and their spatial distribution inside the cell were determined with the aid of intracellularly injected aequorin and an image intensifier-TV system, and cell-to-cell coupling was measured electrically. Treatments with NaCN (5mm), DNP (1.2mm), or ionophore A23187 (2m) caused fall in junctional conductance (uncoupling) that was correlated with [Ca2+]
i
elevation, as was shown before (Rose & Loewenstein, 1976,J. Membrane Biol.
28:87) but not with changes in pH
i
: during the uncoupling induced by CN, the pH
i
(normally 7.5) decreased at most by 0.2 units; during the uncoupling induced by the ionophore, pH
i
fell by 0.13 or rose by 0.3; and in any one of these three agents' uncouplings, the onset of uncoupling and recovery of coupling were out of phase with the changes in pH
i
. Intracellular injection of Ca-citrate or Ca-EGTA solutions buffered to pH 7.2 or 7.5 produced uncoupling with little or no pH
i
change when their free [Ca2+]
i
was >10–5
m. On the other hand, such a solution at pH 4, buffered to [Ca2+]<10–6
m, lowered pH
i
to 6.8 but produced no uncoupling. Thus, a decrease in pH
i
is not necessary for uncoupling in any of these conditions. In fact, uncoupling ensued also during increase in pH
i
: exposure to NH4HCO3 or withdrawal of propionate following exposure to a propionate-containing medium caused pH
i
to rise to 8.74, accompanied by [Ca2+]
i
elevation and uncoupling at pH
i
>7.8.Cell acidification itself can cause elevation of [Ca2+]
i
: injection (iontophoresis) of H+ invariably caused [Ca2+]
i
elevation and uncoupling. These effects were produced also by an application of H+-transporting ionophore Nigericin at extracellular pH 6.5 which caused pH
i
to fall to 6.8. Exposure to 100% CO2 produced a fall in pH
i
, associated in 10 out of 25 cases with [Ca2+]
i
elevation and, invariably, with uncoupling. The absence of a demonstrable [Ca2+]
i
elevation in a proportion of these trials is attributable to depression in Ca2+-measuring sensitivity; inin vivo tests, detection sensitivity for [Ca2+]
i
by aequorin was found to be depressed by the CO2 treatment. Upon CO2 washout, pH
i
and coupling recovered, but onset of recoupling set in at pH
i
as low as 6.32–6.88, generally lower than at the pH
i
at which uncoupling had set in. Exposure to 5% CO2 lowered pH
i
on the average by 0.3 and depressed coupling (in initially poorly coupled cells). After CO2-washout, pH
i
and coupling recovered. During the recovery phase [Ca2+]
i
was elevated, an elevation associated with renewed uncoupling or decrease in rate of recoupling. The results are discussed in connection with possible regulatory mechanisms of junctional permeability. 相似文献
5.
Intracellular Ca2+ (Cai) signaling following the binding of surface receptors activates a Ca2+ permeable plasma membrane conductance which has been shown to be associated with store depletion in a number of cell types. We examined the activation of this conductance in human monocyte-derived macrophages (HMDMs) using whole-cell voltage-clamp techniques coupled with fura-2 microfluorimetry and characterized the importance of external pH (pHo) as a modulator of current amplitude. Current activation was observed following experimental maneuvers designed to deplete intracellular Ca2+-stores including: (i) dialysis of the cell with 100 m inositol 1,4,5-triphosphate (IP3), (ii) intracellular dialysis with high concentrations of the Ca2+ buffers EGTA and BAPTA, or (iii) exposure of the cell to the Ca2+-ATPase inhibitor thapsigargin (1 m). Currents associated with store depletion were inwardly rectifying with kinetics, inactivation, and selectivity that appeared similar irrespective of the mode of activation. Currents were Ca2+ selective with a selectivity sequence of Ca2+ > Sr2+ Mg2+ = Mn2+ = Ni2+. The Ca2+ influx current was modulated by changes in pHo; modulation was not produced as a consequence of changes in internal pH (pHi). External acidification led to a reversible reduction in current amplitude with a pKa at pH 8.2. Changes in pHo alone failed to induce current activation. These observations are consistent with a scheme by which changes in pHo, as would be encountered by macrophages at sites of inflammation, could change the time course and magnitude of the Cai transient associated with receptor activation by regulating the influx of Ca2+ ions.The authors wish to gratefully acknowledge the expert technical assistance of Weiwen Xie without whom the study could not have been completed. This work was supported by National Institutes of Health GM36823. 相似文献
6.
Hubert Felle 《Planta》1988,174(4):495-499
In epidermal cells of maize (Zea mays L.) coleoptiles, cytosolic pH (pHc), cytosolic free calcium, membrane potential and changes thereof were monitored continuously and simultaneously (pHc/,
m, Ca2+/
m) using double-barrelled ion-sensitive microelectrodes. In the resting cells the cytosolic pH was 7.3–7.5 and the concentration of free calcium was 119±24 nM. One-micromolar indole-3-acetic acid (IAA), added to the external medium at pH 6.0 triggered oscillations in
m, pHc and free calcium with a period of 20 to 30 min. Acidification of the cytosolic pH increased the cytosolic free calcium. The
m oscillations are attributed to changes in activity of the H+-extrusion pump at the plasmalemma, triggered off by pH and controlled by pH regulation (pH oscillation). The origin of the pHc and Ca2+ changes remains unclear, but is possibly caused by auxin-receptor-induced lipid breakdown and subsequent second-messenger formation. It is suggested that the observed cytosolic pH and Ca2+ changes are intrinsically interrelated, and it is concluded that this onset of regulatory processes through the phytohormone IAA is indicative of calcium and protons mediating early auxin action in maize coleoptiles. It is further concluded that the double-barrelled ion-sensitive microelectrode is an invaluable tool for investigating in-vivo hormone action in plant tissues.Abbreviations and symbols FC
fusicoccin
- IAA
indole-3-acetic acid
- Mes
2-(N-morpholino)ethanesulfonic acid
- pHc
cytosolic pH
- Tris
2-amino-2-(hydroxymethyl)-1,3-propanediol
-
m
membrane potential difference (mV) 相似文献
7.
Isolated characean internodal cells of Nitellopsis obtusa can be stored in artificial pond water for many days, but they cannot survive in 100mol m?3 NaCl solution unless more than several mol m?3 Ca2+ is added. Short-term effects of NaCl stress on the cytosolic concentration of Ca2+ ([Ca2+]c), cytosolic pH (pHc) and vacuolar pH (pHv) were studied in relation to the external concentration of Ca2+ ([Ca2+]e). Changes in [Ca2+]c were measured with light emission from a Ca2+-sensitive photoprotein, semisynthetic fch-aequorin which had been injected into the cytosol. Both pHc and pHv were measured with double-barrelled pH-sensitive microelectrodes. When internodal cells were treated with 100 mol m?3 NaCl (0–1 mol m?3 NaCl (0.1 mol m?3 [Ca2+]e), [Ca2+]c increased and then recovered to the original level within 60 min. The time course of the transient change in [Ca2+]c was not influenced by the level of [Ca2+]c (0.1 and 10 mol m?3). In some cases, the transient increase in [Ca2+]c was induced only by increasing external osmotic pressure with sorbitol. In response to treatment with 100 mol m?3 NaCl (0.1 mol m?3 [Ca2+]c), pHc decreased by 0.1–0.2 units after 10min but recovered after 30–60 min, while pHv increased by 0.4–0.5 units after 2–50 min and tended to recover after 60 min. The initial changes in both pHc and pHv were suppressed when [Ca2+]e was raised from 0.1 to 10mol m?3. These results show that the charophyte alga Nitellopsis can regulate [Ca2+]c, pHc and pHv under NaCl stress in the short term and that the protective effect of Ca2+ on salinity stress is apparently unrelated to perturbation of Ca2+ and pH homeostasis. 相似文献
8.
Camillo Peracchia 《The Journal of membrane biology》1990,113(1):75-92
Summary Neutral-carrier pH-and Ca-sensitive microelectrodes were used to investigate the relationship between junctional electrical resistance and either pHi or [Ca2+]i in crayfish septate axons uncoupled by acidification. For measuring [Ca2+]i a new neutral carrier sensor sensitive to picomolar [Ca2+] and virtually insensitive to other ions was used. Uncoupling was induced by superfusing the axons with Na-acetate solutions (pH 6.3). With acetate, the time course of changes in junctional resistance differed markedly from that of pHi or [H+]i peaked 40–90 sec before junctional resistance. The difference in shape and peak time between pHi and junctional resistance curves caused significant hysteresis in the pHi versus junctional resistance relationship. In addition, junctional resistance maxima reached with slow acidification rates were 3–4 times greater than those with fast acidifications of similar magnitude. With acetate, [Ca2+]i, increased by approximately one order of magnitude from basal values of 0.1–0.3 m. The curves describing the time course of changes in [Ca2+]i and junctional resistance matched well with each other in shape, peak time and magnitude. Both junctional resistance and [Ca2+]i recovered following a single exponential decay with a time constant of 2 min. Different rates of acidification caused increases in [Ca2+]i and junctional resistance comparable in magnitude. The data indicate that the increase in junctional resistance induced by acidification is more closely related to [Ca2+]i than to [H+]i. 相似文献
9.
Hubert Felle 《Planta》1988,176(2):248-255
In cells of Zea mays (root hairs, coleoptiles) and Riccia fluitans (rhizoids, thalli) intracellular Ca2+ and pH have been measured with double-barrelled microelectrodes. Free Ca2+ activities of 109–187 nM (Riccia rhizoids), 94–160 nM (Riccia thalli), 145–231 nM (Zea root hairs), 84–143 nM (Zea coleoptiles) were found, and therefore identified as cytoplasmic. In a few cases (Riccia rhizoids), free Ca2+ was in the lower millimolar range (2.3±0.8 mM). A change in external Ca2+ from 0.1 to 10 mM caused an initial and short transient increase in cytoplasmic free Ca2+ which finally levelled off at about 0.2 pCa unit below the control, whereas in the presence of cyanide the Ca2+ activity returned to the control level. It is suggested that this behaviour is indicative of active cellular Ca2+ regulation, and since it is energy-dependent, may involve a Ca2+-ATPase. Acidification of the cytoplasmic pH and alkalinization of the vacuolar pH lead to a simultaneous increase in cytoplasmic free Ca2+, while alkalinization of pHc decreased the Ca2+ activity. Since this is true for such remote organisms as Riccia and Zea, it may be concluded that regulation of cytoplasmic pH and free Ca2+ are interrelated. It is further concluded that double-barrelled microelectrodes are useful tools for investigations of intracellular ion activities in plant cells.Symbols and abbreviations m, m
membrane potential difference, changes thereof
- PVC
polyvinylchloride 相似文献
10.
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
11.
The effects of D-glucose, D-glyceraldehyde, glibenclamide, D-600, NH
4
+
and high concentrations of K+ on cytoplasmic pH (pH
i
) were investigated in dispersed and cultured pancreatic -cells fromob/ob mice. The cytoplasmic pH was measured with the fluorescent H+-indicator quene 1. The average pH
i
value in resting -cells was 6.71. Addition of 20 mM of the physiological stimulus D-glucose increased pH
i
with 0.05 units. Both glibenclamide and high concentrations of K+ decreased pH
i
. The latter effects were completely reversed by D-600, supporting the notion that free cytoplasmic Ca2+ can be involved in the regulation of pH
i
. In contrast to D-glucose, 10mM of D-glyceraldehyde decreased pH
i
by 0.09 units, an effect persisting even in the presence of D-600. From the present study it is evident that D-glyceraldehyde and D-glucose have opposite effects on pH
i
in pancreatic -cells. 相似文献
12.
The transition of a resting macrophage into the activated state is accompanied by changes in membrane potential, cytoplasmic pH, and intracellular calcium (Ca
i
). Activation of Cl– as well as H+-selective currents may give rise to stimulus-induced changes in membrane potential and counteract changes in intra-cellular pH (pH
i
) which have been observed to be closely associated with respiratory burst activation and superoxide production in macrophages. We carried out whole-cell voltage clamp experiments on human monocyte-derived macrophages (HMDMs) and characterized currents activated following an elevation in Ca
i
using isosmotic pipette and bath solutions in which Cl– was the major permeant species. Ca
i
was elevated by exposing cells to the Ca2+ ionophore A23187 (1–10 m) in the presence of extracellular Ca2+ or by internally exchanging the patch-electrode solution with ones buffered to free Ca2+ concentrations between 40 and 2,000 nm. We have identified two Ca2+-dependent ion conductances based on differences in their characteristic time-dependent kinetics: a rapidly activating Cl– conductance that showed variable inactivation at depolarized potentials and a H+ conductance with delayed activation kinetics. Both conductances were inhibited by the disulfonic acid stilbene DIDS (100 m). Current activation for both Ca2+-dependent conductances was phosphorylation dependent, neither conductance appeared in the presence of the broad spectrum kinase inhibitor H-7 (75 m). Inclusion of the autophosphorylated, Ca2+/calmodulin-dependent protein kinase in the pipette in the presence of ATP induced a rapidly activating current similar to that observed following an elevation in Ca
i
. Activation of both conductances would contribute to the changes in membrane potential which accompany stimulation-induced activation of macrophages as well as counteract the decrease in pH
i
during sustained Superoxide production.The authors wish to thank Dr. H. Schulman for providing us with the purified CaMKII and Jennifer Foss for technical assistance. This work was supported by National Institutes of Health RO1 GM36823. 相似文献
13.
Otto Baumann Bernd Walz 《Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology》1989,165(5):627-636
Summary The photoreceptor cells in the honeybee drone contain an elaborate Ca2+-sequestering endoplasmic reticulum (ER). We measured Ca-oxalate formation within the ER of permeabilized retinal slices with a microphotometer and studied the kinetics of Ca2+-uptake into the ER and the properties of Ins(1,4,5)P3-induced Ca2+-release.The ATP-dependent Ca2+-uptake mechanism has a high affinity for Ca2+: Uptake rate was half maximal at Ca2+
free 0.6 M.Addition of Ins(1,4,5)P3 caused a persistent depression of Ca-oxalate formation due to Ca2+ -release from the ER. The Ins(1,4,5)P3-dependent Ca2+-release mechanism has a high affinity (half maximal rate with 0.2 M Ins(1,4,5)P3) and a high specificity for Ins(1,4,5)P3: Ins(2,4,5)P3 was 6 times, Ins(1,3,4,5)P4 was 15 times less potent in inducing Ca2+-release. 3 M Ins(1,4)P2 had no detectable effect. The sensitivity for Ins(1,4,5)P3 was maximal between 280 nM and 1.6 M Ca2+
free and decreased at higher and lower Ca2+-concentrations.Our data show that the ER in invertebrate photoreceptor cells is an effective Ca2+ -sink and an Ins(1,4,5)P3-sensitive Ca2+-source. We support the idea (Payne et al. 1988) that the ER-network close to the photoreceptive membrane, the submicrovillar cisternae (SMC), are the light- and Ins(1,4,5)P3-sensitive Ca2+-stores.Abbreviations
ER
endoplasmic reticulum
-
Ins(1,4,5)P
3
D-inositol 1,4,5-trisphosphate
-
Ins(1,3,4)P
3
D-inositol 1,3,4-trisphosphate
-
Ins(2,4,5)P
3
D-inositol 2,4,5-trisphosphate
-
Ins(1,4)P
2
D-inositol 1,4-bisphosphate
-
Ins(1,3,4,5)P
4
D-inositol 1,3,4,5-tetrakisphosphate
-
SMC
submicrovillar cisternae
-
[Ca
2+]i
intracellular free Ca2+-concentration 相似文献
14.
Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na+/K+-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pHi), intracellular Ca2+ ([Ca2+]i) and Na+/K+-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca2+ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 M) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca2+]i (200%). A decrease in pHi (from 7.3 ± 0.09 to 6.4 ± 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca2+. Inhibition of Na+/H2+ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca2+ using CCCP (10 M) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K+/H+ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca2+ influx via Na+/Ca2+ exchange, which under hyposmotic conditions activates the K+ and Ca2+ mitochondrial exchange systems (K+/H+ and Ca2+/H+).Deceased 相似文献
15.
Using a Ca2+-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K+]
e
up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca2+]
i
, whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA2+-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca2+]
i
and a decrease in the K+-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca2+]
i
. Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca2+ influx and Ca2+-induced Ca2+ release from the endoplasmic reticulum; the initial level of [Ca2+]
i
was restored at the joint involvement of Ca2+-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria. 相似文献
16.
The Ca2+-extruding ATPase pump of the human platelet was studiedin situ by measuring Ca2+ extrusion from quin2-overloaded platelets (Johansson, J.S., Haynes, D.H. 1988.J. Membrane Biol.
104:147–163). Cytoplasmic pH (pHcyt) was measured by BCECF fluorescence in parallel experiments. The pump was studied by raising the cytoplasmic free Ca2+ to 2.5 μM and monitoring active Ca2+ extrusion into a Ca2+-free medium. The pump was shown to perturb pHcyt, to not respond to changes in membrane potential and to respond to imposed changes in pHcyt in a manner consistent with the Ca2+ pump acting as a 2 Ca2+/nH+ exchanger. (i) Raising the external pH (pHext) from 7.40 to 7.60 lowers the Vmax of the pump in basal condition (Vmax,1) from 110±18 to 73±12 μM/min (=μmol/liter cell volume/min). (ii) Lowering pHext to 7.13 raised Vmax,1 to 150±15 μM/min. (iii) In an N-methyl-d-glucamine (NMDG+) medium, the pump operation against high [Ca2+]cyt acidifies the cytoplasm by −0.36±0.10 pH units, and the pump becomes self-inhibited. (iv) Use of nigericin to drive pHcyt down to 6.23 reduces the Vmax,1 to 18±11 μM/min. (v) Alkalinization of the cytoplasm by monensin in the presence of Na+ raises the Vmax,1 (basal state withK
m,1=80 nM) to 136±24 μM/min, but also activates the pump fourfold (Vmax,2=280±28 μM/min;K
m,2=502±36 nM). (vi) Transient elevation of pHcyt by NH4Cl at high [Ca2+]cyt activates the pump eightfold (Vmax,2≥671±350 μM/min). The large activation by alkaline pHcyt at high [Ca2+]cyt can be explained by Ca2+-calmodulin activation of the pump (Valant, P.A., Adjei, P.N., Haynes, D.H. 1992.J. Membrane Biol.
130:63–82) and by increased Ca2+ affinity of calmodulin at high pH. 相似文献
17.
Parvathi K. Gayathri J. Maralihalli G.B. Bhagwat A.S. Raghavendra A.S. 《Photosynthetica》2000,38(1):23-28
PEP carboxylase (PEPC) in leaves of C4 plants is activated by phosphorylation of enzyme by a PEPC-protein kinase (PEPC-PK). We reevaluated the pattern of PEPC phosphorylation in leaf extracts of Amaranthus hypochondriacus. It was dependent on Ca2+, the optimum concentration of which for stimulation was 10 mM. The extent of stimulation was inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), a Ca2+ chelator. The inhibition by BAPTA was relieved by the addition of Ca2+ but not by the addition of Mg2+. The stimulation by Ca2+ of PEPC phosphorylation was marginally enhanced by calmodulin (CaM), but not by diacylglycerol (DAG). Phosphorylation was strongly restricted by Ca2+ or Ca2+-CaM-dependent protein kinase inhibitors. Thus phosphorylation of PEPC is Ca2+-dependent in leaves of A. hypochondriacus and a calcium-dependent protein kinase (CDPK) may modulate PEPC-PK and subsequently the phosphorylation status of PEPC. 相似文献
18.
Regulatory relationship and gain control between cytosolic free Ca2+ concentration (Cai) and cytosolic pH (pHi) were evaluated by two different cell types, gastric parietal cells, and blood platelets. Studies were carried out in both single cells and populations of cells, using Ca2+-indicative probe fura-2 (1-(2-(5′-carboxyoxazol-2′-yl)-6-aminobenzofuran-5-oxy)-2-(2′-amino-5′-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid) and pH-indicative probe BCECF (2′,7′-bis(carboxyethyl) carboxyfluorescein). Stimulation of single and populational parietal cells and platelets with gastrin and thrombin, respectively, resulted in an increase in Cai. In both populational cell types, an initial change in pHi during agonist stimulation occurred almost simultaneously with the mobilization of Ca2+; an initial transient decrease in pHi was followed by a slower increase in pHi above the prestimulation level. When populational platelets were preloaded with the Ca2+ chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′tetraacetic acid), the thrombin-induced initial large increase in Cai was apparently inhibited, whereas the pHi decrease induced by thrombin was not altered. This suggests that the initial Cai change is not a prerequisite for the pHi change. The effect of pHi on Cai was examined next. In both single and populational cell types, application of the K+-H+ ionophore nigericin, which induced a transient decrease in pHi, led to the release of Ca2+ from intracellular stores. In single parietal cells double-labeled with fura-2 and BCECF, a temporal decrease in pHi preceded the rise in Cai after stimulation with nigericin. A decrease in pHi, and an increase in Cai occurred at 1.5 and 4 s, respectively. In single parietal cells, replacement of medium Na+ with N-methyl-
-glucamine (NMG+), which also induced a decrease in pHi, resulted in repetitive Ca2+ spike oscillations. The source of Ca2+ utilized for the Ca2+ oscillation that was induced by NMG+ originated from the agonist-sensitive pool. Thus, several maneuvers, which were capable of decreasing pHi, led to an increase in Cai. Cytosolic acidification may be a part of the trigger for Ca2+ mobilization from intracellular stores in both parietal cells and platelets. 相似文献
19.
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
20.
Summary Calcium signaling systems in nonexcitable cells involve activation of Ca2+ entry across the plasma membrane and release from intracellular stores as well as activation of Ca2+ pumps and inhibition of passive Ca2+ pathways to ensure exact regulation of free cytosolic Ca2+ concentration ([Ca2+]
i
). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca2+ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca2+ entry and release while bradykinin appeared only to release Ca2+ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca2+]
i
response to these agonists was examined by four methods. Low concentrations of TPA (2×10–10
m) had no effect on Ca2+ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca2+]
i
to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca2+]
i
changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca2+ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca2+ entry, fura-2 loaded cells were suspended in Mn2+ rather than Ca2+ buffers. Addition of EGF or TGF- resulted in Ca2+ release and Mn2+ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn2+ influx. Thus, PKC is able to regulate Ca2+ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10–8
m) inhibited not only Ca2+ entry but also Ca2+ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca2+ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca2+]
i
, showing a common pool of Ca2+ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca2+]
i
transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca2+ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca2+]
i
signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca2+ for EGF-mediated Ca2+ release which also respond differently to TPA. 相似文献