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1.
Summary Homeostasis of intracellular calcium ([Ca++]i) and pH (pHi) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain
normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes
of [Ca++]i and pHi on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure
[Ca++]i with fura-2 and pHi with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pHi in these cells was 7.37±0.05 (n=20 cells) and resting [Ca++]i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH4Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca++]i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH4Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium
stores. On removal of NH4Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pHi decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca++]i, but produced a biphasic change in pHi. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial
values. When [Ca++]i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pHi decreased by 0.35±0.02 units. We conclude that an increase in pHi leads to an increase in [Ca++]i in rabbit corneal epithelial cells; however, a decrease in pHi leads to minor changes in [Ca++]i. The ability of CE cells to maintain proper calcium homeostasis when pHi is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification,
which occur during prolonged eye closure. 相似文献
2.
E. Edward Bittar Bo G. Danielson Warren Lin John Richards 《The Journal of membrane biology》1977,34(1):223-246
Summary Radiosodium efflux from barnacle muscle fibers is a function of pH
e
, the threshold pH
e
for stimulation of Na efflux into HCO
3
–
-artificial sea water (ASW) being 6.8 and the fixed thresholdpCO2 (in an open CO2 system) being approximately 30 mm Hg. Acidification of ASW containing non-HCO
3
–
buffer is without effect on the Na efflux. The Na efflux following stimulation by reducing the pH of 10mM HCO
3
–
-ASW from 7.8 to 6.3 is reduced by 17.3% as the result of microinjecting 100mM EGTA, and increased by 32.6% as the result of microinjecting 0.5M ATP. The Na efflux into K-free HCO
3
–
-ASW is markedly stimulated by external acidification. Ouabain-poisoned fibers are more responsive to a low pH
e
than unpoisoned fibers. Applying the 2-14C-DMO technique, it is found that fibers bathed in 10mM HCO
3
–
-ASW at pH 7.8 have an internal pH of 7.09±0.106 (mean±SD), whereas fibers bathed in 25mM TRIS-ASW at pH 7.8 have a pH
i
of 7.28±0.112. The relationship between pH
i
and pH
e
as external pH is varied by adding H+ is linear. Measurements of the resting membrane potential indicate that external acidification in the presence of HCO
3
–
as buffer is accompanied by a fall inE
m
, the threshold pH
e
being 7.3 both at 24 and 0°C. This sensitivity amounts to 8.2 mV per pH unit (at 24°C) over a wide range of pH
e
. Membrane resistance following external acidification remains unchanged. Microinjection of the protein inhibitor of Walsh before external acidification fails to stop depolarization from occurring. Cooling to 0°C also fails to abolish depolarization following acidification. Whereas external ouabain and ethacrynic acid reduceE
m
in the absence or presence of acidification, DPH hyperpolarizes the membrane or arrests depolarization both at 24 and 0°C. This effect of DPH at 0°C is seen in the absence or presence of acidification. It is suggested that depolarization following acidification of a HCO
3
–
-containing medium is due to activation of a Cl–-and/or HCO
3
–
-pump and that ouabain and ethacrynic acid reducesE
m
by abolishing uncoupled Na transport. 相似文献
3.
Accumulation of products of lipid peroxidation (malondialdehyde, conjugated dienes, lipid peroxides, and Schiff bases) was evaluated in rabbit kidney cortex slices made ischemic for 60 min followed by 18 h storage at 5°C in UW Na gluconate solution and 210 min normothermic reoxygenated incubation. In addition, the effect of adding Trolox (1 mM), deferoxamine (1 mM), and ascorbate (1 mM) as supplemental antioxidants to the UW gluconate solution was evaluated. Lipid peroxidation was slightly increased after hypothermic storage compared to slices subjected to ischemia alone but was not significantly different than ischemic slices during subsequent incubation at normothermia. The addition of either deferoxamine or Trolox to the storage solution substantially reduced lipid peroxidation both during hypothermic storage and subsequent to normothermic incubation. Ascorbate had a mild prooxidant effect as a sole additive to the UW gluconate solution but was clearly prooxidant when combined with either deferoxamine or Trolox. These results suggest that supplemental antioxidants added to the UW gluconate solution under conditions analogous to machine perfusion preservation have a potential role in reducing oxidative stress in kidney tissues harvested after warm ischemia and that hypothermia may be a valuable adjunct to resuscitative therapeutic regimens developed for salvage of ischemic kidneys for transplantation. 相似文献
4.
Complexation of M+=Li+, Na+, Ag+ and TI+ by the cryptands 4, 7, 13, 18-tetraoxa-l, 10-diazabicyclo[8.5.5]eicosane (C211) and 4,7,13-trioxa-1,10-diazabicyclo[8.5.5]eicosane (C21C5) to form the cryptates [M.C211]+ and [M.C21C5]+ has been studied in trimethyl phosphate by potentiometric titration and 7Li and 23Na NMR spectroscopy. For [M.C211]+ the logarithm of the apparent stability constants, log K (dm3 mol-1)=6.98±0.05, 5.38±0.05, 9.82±0.02 and 3.95±0.02 for M+ =Li+, Na+, Ag+ and TI+, respectively; and for [M.C21C5]+ log K (dm3 mol-1)=2.40±0.10, 1.90±0.05, 6.04±0.02 and 2.42±0.10 for M+=Li+, Na+, Ag+ and Tl+, respectively. The decomplexation kinetic parameters for [Na.C211]+ are: kd (298.2 K)=6.924±0.50 s-l, ΔHd≠=62.2±0.9 kJ mol-1, and ΔSd≠= -20.3±2.7 J K-1 mol-1; and those for [Li.C21C5]+ are: kd (298.2 K)=23.3±0.4 s-1, ΔHd≠ =61.2±1.1 kJ mol-1, and ΔSd≠= -13.6±3.6 J K-1 mol-1. Metal ion exchange on [Li.C211]+ is in the very slow extreme of the NMR timescale up to 390 K and kd « 4 s-1 at 298.2 K, while in contrast exchange on [Na.C21C5]+ is in the fast extreme of the NMR timescale at 298.2 K (kd≈ 104 s-1). These data are compared with those obtained in other solvents. 相似文献
5.
The H+/ATP synthase from yeast mitochondria, MF0F1, was purified and reconstituted into liposomes prepared from phosphatidylcholine and phosphatidic acid. Analysis by mass spectrometry revealed the presence of all subunits of the yeast enzyme with the exception of the K-subunit. The MF0F1 liposomes were energized by acid-base transitions (ΔpH) and a K+/valinomycin diffusion potential (Δφ). ATP synthesis was completely abolished by the addition of uncouplers as well as by the inhibitor oligomycin. The rate of ATP synthesis was optimized as a function of various parameters and reached a maximum value (turnover number) of 120 s− 1 at a transmembrane pH difference of 3.2 units (at pHin = 4.8 and pHout = 8.0) and a Δφ of 133 mV (Nernst potential). Functional studies showed that the monomeric MF0F1 was fully active in ATP synthesis. The turnover increased in a sigmoidal way with increasing internal and decreasing external proton concentration. The dependence of the turnover on the phosphate concentration and the dependence of KM on pHout indicated that the substrate for ATP synthesis is the monoanionic phosphate species H2PO4−. 相似文献
6.
Summary H+-coupled transport in plant and fungal cells is relatively insensitive to external pH (pH
o
). H+-coupled Cl– transport at the plasma membrane ofChara corallina was studied to explore the phenomena responsible for this insensitivity. Raising pH
o
from a control value of 7.5 to 9.0 results in a modest (2.5-fold) decline inJ
max and increase inK
m
. Further increase in pH
o
results in a selective increase inJ
max, in accordance with predictions from a reaction kinetic model of the transport system (Sanders, D., Hansen, U.-P., 1981.J. Membrane Biol.
58:139–153). Increase in cytosolic Cl– concentration ([Cl–]
c
) also results in a selective decrease inJ
max at pH
o
=7.5.Quantitative kinetic modeling of the results is not possible if it is assumed that the sole effect of pH
o
isvia mass action on the binding of external H+ to a transport site. If, instead, the dependence of cytosolic pH (pH
c
) on pH
o
(Smith, F.A., 1984,J. Exp. Bot.
35:1525–1536) is taken into account along with the dependence of Cl– influx on pH
c
(Sanders, D., 1980,J. Membrane Biol.
53:129–141), then the observed modest changes in Michaelis parameters can be accommodated by a reaction kinetic model. The quantitative parameters of the model yield respective pK
a
s of the internal and external H+-binding sites=7.85 and 7.2, respective dissociation constants of the internal and external Cl–-binding sites=160 and 40 m, and an additional, kinetically transparent, H+-binding site with a pK
a
>8.0. The quantitative model independently predicts the response ofJ
max andK
m
to acidic conditions.The results are discussed in terms of the general physiological requirement that fluxes through H+-coupled transport systems are relatively insensitive to environmental variation in pH
o
. It is proposed that (i) the weak (but finite) dependence of pH
c
on pH
o
, coupled with (ii) the strong dependence of H+-coupled transport on pH
c
are instrumental in endowing H+-coupled transport systems with a relative insensitivity to variation in pH
o
. This hypothesis might also explain why pH
c
in plants and fungi is not acutely controlled with respect to variation of pH
o
. 相似文献
7.
Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H+ gradient (pHin=6.0, pHout=8.0)22Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pHin=pHout=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K
1
=1.6 m) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K+-stimulatedp-nitrophenyl phosphatase. In addition22Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H+ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK
M
=8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na+/H+ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion. 相似文献
8.
Charles M. Schron 《The Journal of membrane biology》1991,120(2):192-200
Summary In jejunal brush-border membrane vesicles, an outwardly directed OH– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M. 1985.J. Clin. Invest.
76:2030–2033), suggesting carrier-mediated folate: OH– exchange (or phenomenologically indistinguishable H+: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examining F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was greater at lower (pHint/pHext: 5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive3H-folate uptake only at pH6.0. Since stepwise increments ininternal pH (4.57.5; pHext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH (4.356.5; pHint=7.8), a 50-fold decrement in F uptake was observed (H+
K
m
=12.8±1.2 M). Hill plots of these data suggest involvement of at least one H+ (OH–) at low pH (monovalent F– predominates) and at least 2 H+ (OH–) at high pH (divalent F–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pHext 4.55 or 5.8, transport of F– and F–2 is electroneutral. Kinetic parameters for F– and F–2 were calculated from uptake data at pHext 4.55 and 5.0. Comparison of predictedvs. experimentally determined kinetic parameters at pHext5.8 (K
m
=1.33vs. 1.70 M;V
max=123.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV
max, but does not affect theK
m
for carrier-mediated F transport. These data are consistent with similarK
i
'
s for sulfasalazine (competitive inhibitor) at pHext 5.35 and 5.8 (64.7 and 58.5 M, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalent F and is sensitive to external pH with a H+
K
m
(or OH– lC50) corresponding to pH 4.89. External pH effects theV
max, but not theK
m
for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH– or H+),rather than competitive binding that is mutually exclusive. 相似文献
9.
Data are presented regarding the establishment of the pH (designated pH*) of a standard buffer solution suitable as a pH reference in 50 mass% glycerol/water mixtures at temperatures ranging from −20 to 25 °C. The buffer material selected was the ampholyte Mops [(3-N-morpholino)-propane sulfonic acid], and the reference standard consists of equal molal amounts of Mops and its sodium salt. The assignment of pH* values is based on measurements of the electromotive force (emf) of cells without liquid junction of the type: Pt;H2(g,1 atm) ¦ Mops, Na Mopsate, NaCl ¦ AgCl;Ag and the pH* was derived from a determination of K2, the equilibrium constant for the dissociation process (Mops)±/ah (Mopsate)− + H +. The standard emf of the silver-silver chloride electrode in 30, 40, and 50 mass% glycerol/water mixtures was determined from emf measurements of the cell at subzero temperatures with HCl solutions replacing the buffer-chloride mixtures. 相似文献
10.
Terutaka Ozawa Yoshitaka Saito Akinori Nishiyama 《The Journal of membrane biology》1988,105(3):273-280
Summary Anion exchange transport in the mouse lacrimal gland acinar cell membrane was studied by measuring the intracellular H+ (pHi) and Cl– (aCli) activities with double-barreled ion-selective microelectrodes. In a HCO
3
–
-free solution of pH 7.4 (HEPES/Tris buffered), pHi was 7.25 andaCli was 33mm. By an exposure to a HCO
3
–
(25mm HCO
3
–
/5% CO2, pH 7.4) solution for 15 min,aCli was decreased to 25mm and pHi was transiently decreased to about 7.05 within 1 min, then slowly relaxed to 7.18 in 15 min. Intracellular HCO
3
–
concentration [HCO
3
–
]i, calculated by the Henderson-Hasselbalch's equation, was 11mm at 1 min after the exposure and then slowly increased to 15mm. Readmission of the HCO
3
–
-free solution reversed the changes inaCli and pHi. The intracellular buffering power was about 40mm/pH. An addition of DIDS (0.2mm) significantly inhibited the rates of change inaCli, pHi, and [HCO
3
–
]i caused by admission/withdrawal of the HCO
3
–
, solution and decreased the buffer value. Replacement of all Cl– with gluconate in the HCO
3
–
solution increased pHi, and readmission of Cl– decreased pHi. The rates of these changes in pHi were reduced by DIDS by 32–45% but not by amiloride (0.3mm). In the HCO
3
–
solution, a stimulation of intracellular HCO
3
–
production by exposing the tissue to 25mm NH
4
+
increasedaCli significantly. While in the HCO
3
–
-free solution or in the HCO
3
–
, solution containing DIDS, exposure to NH
4
+
had little effect onaCli. All of these findings were consistent with the presence of a reversible, disulfonic stilbene-sensitive Cl–/HCO
3
–
exchanger in the basolateral membrane of the acinar cells. The possibility of anion antiport different from one-for-one Cl–/HCO
3
–
exchange is discussed. 相似文献
11.
Mamun M. Or-Rashid Ryoji Onodera Shaila Wadud Nazimuddin Mohammed 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2000,741(2)
A high-performance liquid chromatographic procedure for the quantitative determination of cysteine (Cys), homocysteine (Hcys), methionine sulfoxide (MSO), methionine sulfone (MSO2), homoserine (Hser), glycine (Gly), threonine (Thr), 2-aminobutyric acid (2AB), methionine (Met), cystathionine (Cysta) and its application to rumen fluid are described. The samples containing Thr, Met and other related amino compounds were derivatized with 9-fluorenylmethyl chloroformate. The separation of compounds was accomplished with a methanol gradient in 25 mM sodium citrate buffer (obtaining pH 6.40 and 3.80 by addition of 25 mM citric acid). All derivatized compounds were separated on a Mightysil RP-18 GP (150×4.6 mm I.D., 5 μm particle size) column. All analytes were detected at 265 nm with UV detection. The limits of detection (μM) (S/N ratio, 3:1) and quantification (μM) (S/N ratio, 10:1) of Cys, Hcys, MSO, MSO2, Hser, Gly, Thr, 2AB, Met and Cysta were 0.50 and 1.68; 1.76 and 5.85; 0.85 and 2.88; 0.92 and 3.09; 1.04 and 3.52; 0.76 and 2.52; 0.65 and 2.18; 0.39 and 1.36; 0.31 and 1.03; 0.17 and 0.58, respectively. The recoveries of all compounds in rumen fluid were 97.93–102.3% in the within-day study and 94.52–98.69% on different day (6 days) studies. The average contents (μM) of Cys, Gly, Thr, 2AB, Met and Cysta were 1.72, 45.6, 20.0, 4.3, 2.11 and 3.42 before morning feeding. The concentration of Thr, 2AB and Cysta in rumen fluid tended to increase with time after feeding whereas Met showed the opposite tendency. 相似文献
12.
Charles M. Schron 《The Journal of membrane biology》1990,118(3):259-267
Summary In jejunal brush-border membrane vesicles, an out-wardly directed OH– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M., 1985).J. Clin. Invest.
76:2030–2033), suggesting carrier-mediated folate: OH– exchange (or phenomenologically indistiguishable H+: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examinin F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was geater at lower (pHint/pHext:5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive3H-folate uptake only at pH6.0. Since setepwise increments ininternal pH (4.57.5; pHext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH(4.356.5; pHint=7.8), a 50-fold decrement in F uptake was observed (H+
K
m
=12.8±1.2m). Hill plots of these data suggest involvement of at least one H+ (OH–) at high pH (divalent F–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pHext 4.55 or 5.8, transport of F– and F–2 is electroneutral. Kinetic parameters for F– and F–2 were calculated from uptake data at pHext 4.55 and 5.0. Comparision of predictedvs. experimentally determined kinetic parameters at pHext 5.8 (K
m
=1.33vs. 1.70 m;V
max=12.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV
max, but does not affect thatK
m, for carrier-mediated F transport. These data are consistent with similarK
i's for sulfasalazine (competitive inhibitor) at pHext 5.35 and 5.8 (64.7 and 58.5 m, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalen F and is sensitive to extermal pH with a H+
K
m
(or OH– IC50) corresponding to pH 4.89. External pH affects theV
max, but not theK
m
for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH– or H+) rather than competitive binding that is mutually exclusive. 相似文献
13.
Randall K Packer Jeffrey L Garvin 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1998,120(4):777-783
We measured Na+/K+ ATPase activity in homogenates of gill tissue prepared from field caught, winter and summer acclimatized yellow perch, Perca flavescens. Water temperatures were 2–4°C in winter and 19–22°C in summer. Na+/K+ ATPase activity was measured at 8, 17, 25, and 37°C. Vmax values for winter fish increased from 0.48±0.07 μmol P mg−1 protein h−1 at 8°C to 7.21±0.79 μmol P mg−1 protein h−1 at 37°C. In summer fish it ranged from 0.46±0.08 (8°C) to 3.86±0.50 (37°C) μmol P mg−1 protein h−1. The Km for ATP and for Na+ at 8°C was ≈1.6 and 10 mM, respectively and did not vary significantly with assay temperature in homogenates from summer fish. The activation energy for Na+/K+ ATPase from summer fish was 10 309 (μmol P mg−1 h−1) K−1. In winter fish, the Km for ATP and Na+ increased from 0.59±0.08 mM and 9.56±1.18 mM at 8°C to 1.49±0.11 and 17.88±2.64 mM at 17°C. The Km values for ATP and Na did not vary from 17 to 37°C. A single activation energy could not be calculated for Na/K ATPase from winter fish. The observed differences in enzyme activities and affinities could be due to seasonal changes in membrane lipids, differences in the amount of enzyme, or changes in isozyme expression. 相似文献
14.
Summary The intracellular pH (pH
i
) of Ehrlich ascites tumor cells, both in the steady state and under conditions of acid loading or recovery from acid loading, was investigated by measuring the transmembrane flux of H+ equivalents and correlating this with changes in the distribution ratio of dimethyloxazolidine-2,4-dione (DMO). The pH
i
of cells placed in an acidic medium (pH
o
below 7.15) decreases and reaches a steady-state value that is more alkaline than the outside. For example when pH
o
is acutely reduced to 5.5, pH
i
falls exponentially from 7.20 ± 0.06 to 6.29 ± 0.04 with a halftime of 5.92 ± 1.37 min, suggesting a rapid influx of H+. The unidirectional influx of H+ exhibits saturation kinetics with respect to extracellular [H+]; the maximal flux is 15.8 ± 0.05 mmol/(kg dry wt · min) andK
m
is 0.74 ± 0.09 × 10–6
m.Steady-state cells with pH
i
above 6.8 continuously extrude H+ by a process that is not dependent on ATP but is inhibited by anaerobiosis. Acid-loaded cells (pH
i
6.3) when returned to pH
o
7.3 medium respond by transporting H+, resulting in a rapid rise in pH
i
. The halftime for this process is 1.09 ± 0.22 min. The H+ efflux measured under similar conditions increases as the intracellular acid load increases. An ATP-independent as well as an ATP-dependent efflux contributes to the restoration of pH
i
to its steady-state value. 相似文献
15.
Organ preservation solutions have been designed to protect grafts against the injury inflicted by cold ischemia. However, toxicity of University of Wisconsin (UW) solution during rewarming has been reported. Therefore, we here assessed the toxicity of UW, histidine-tryptophan-ketoglutarate (HTK), Euro-Collins, histidine-lactobionate (HL), sodium-lactobionate-sucrose and Celsior solutions in cultured hepatocytes under hypothermic (4 °C), intermediate (21 °C) and physiological (37 °C) conditions. Marked toxicity of UW, HTK, HL and Euro-Collins solutions was observed at both 37 and 21 °C. With the exception of UW solution, these solutions also increased cell injury during cold incubation (LDH release after 18 h at 4 °C: HTK 76 ± 2%, Euro-Collins 78 ± 17%, HL 81 ± 15%; control: Krebs-Henseleit buffer 20 ± 6%). Testing of individual components using modified Krebs-Henseleit buffers suggested that histidine and phosphate are responsible for (part of) this toxicity. These potential toxicities should be taken into account in the development of future preservation solutions. 相似文献
16.
E. García-Martín F. J. Martín-Romero C. Gutiérrez-Merino 《Journal of neurochemistry》1995,65(6):2757-2764
Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg2+ concentration ([Mg2+]i) was found to be dependent on external Mg2+ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg2+ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na+ gradient across the plasma membrane of synaptosomes by treatment with the Na+ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg2+]i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg2+. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg2+]i. Reversal of the Na+ gradient by incubation of synaptosomes in a medium in which external Na+ was replaced by choline increased [Mg2+]i up to 1.6 and 2.2 mM for extrasynaptosomal Mg2+ concentrations of 1 and 8 mM, respectively. We conclude that a Na+/Mg2+ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg2+ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg2+]i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg2+]i. It is concluded that Mg2+ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase. 相似文献
17.
An analog of adenosine 5′-triphosphate (ATP) was synthesized in which the C4′---C5′---O---Pα system is replaced by a trans C4′---CH=CH---Pα system. In the form of 1:1 complexes with Mg, this analog and its counterpart with a C4′---CH2---CH2---Pα system were linear competitive inhibitors, with respect to MgATP, of the MAT-II (normal tissue) and MAT-T (hepatoma tissue) forms of rat ATP:
-methionine-S-adenosyltransferase (MAT); Km(ATP)/Ki values ranged from 0.4 to 2.4. 2′-Deoxy-ATP was a weak substrate, Km(ATP)/Km = 0.035, of MAT-II and a weak competitive inhibitor, Km(ATP)/Ki = 0.07, of MAT-T. These findings, together with interactions of the MAT forms with other substrates and inhibitors, indicate that binding of ATP to these transferases is accompanied by little rotation about the C5′---O5′ bond, and that C4′ and Pα are in a trans-type relationship in enzyme-bound ATP. 相似文献
18.
N. Vaysse J. Laval C. Senarens J.P. Esteve A. Ribet 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1982,720(4)
Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [3H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10−8 M and was half-maximal at 7.9±3.4·10−7M. The increase at 1·10−5 M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10−9 M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC50 value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10−5 M dopamine was 2.3±0.9·10−6M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [3H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The Kd value for high affinity binding sites was 0.43±0.1·10−7M and 4.7±1.6·10−7M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10/t-7M noradrenaline, 2·10/t-7 M epinine, 4.1±1.8·10/t-6M fluphenazine, 8.0±1.6·10/t-6M haloperidol, 4.2±1.2·10−6Mcis-flupenthixol, 2.7±0.4·10−5Mtrans-flupenthixol, >1·10−5M apomorphine, sulpiride, naloxone and isoproterenol. 相似文献
19.
The dependence of the membrane potential (Em) and the membrane resistance (Rm) of Chara australis R. Brown on the pH of the external medium (pH0) was studied by controlling the activity of the plasmamembrane H+ pump under both light and dark conditions. The activity of the pump was controlled by regulating the internal ATP or Mg2+ concentration in tonoplast-free cells prepared by vacuolar perfusion. In these cells, which contained Mg · ATP (mgATP cells), Em and Rm were very sensitive to pH0, as in normal cells. Em was more negative in light than in the dark at all pH0 values tested. Tonoplast-free cells with very low [ATP]i (-ATP cells) or [Mg2+]i (-Mg cells) showed very weak dependence of Em and Rm on pH0. Thus, the active and not the passive component of Em was sensitive to pH0. At the same time, the high permeability of the plasma membrane to H+ was questioned. In both-ATP cells and-Mg cells, Em was scarcely affected and Rm markedly decreased on illumination.Abbreviations CyDTA
1,2-cyclohexanediamine-N,N-tetraacetic acid
- EGTA
ethyleneglycol-bis-(-aminoethylether)N,N-tetraacetic acid
- HK
hexokinase 相似文献
20.
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