共查询到20条相似文献,搜索用时 15 毫秒
1.
Hiroshi Tanaka Yumiko Ohta Tsuneaki Sugimoto Yoshihisa Kurachi 《The Journal of membrane biology》1991,120(2):185-190
Summary The electrical properties of a mouse interleukin (IL)-3-dependent cell line, FDC-P2, were examined using the tightseal whole-cell clamp technique. Under current clamp conditions with 140mM K+ in the pipette, the cells had a resting potential of –30 mV. Under voltage-clamp conditions, a transient outward current was elicited upon depolarization from a holding potential of –80 mV. The current was activated at potentials more positive than –10 mV and had a delayed-rectifying property. It showed rapid activation and slow inactivation during command steps. The current was abolished by Cs+ in the pipette, indicating that K+ is the charge carrier. The K+ current was suppressed by tetraethylammonium withK
i
of <0.1mM and was not affected by scorpion toxin. Recovery from inactivation was steeply voltage dependent: As the holding potential was more hyperpolarized, the recovery became faster. Thus, with a holding potential of –80 mV, the current showed slight use-dependent inactivation, while the current decreased prominently by repetitive depolarization at a holding potential of –40 mV. These properties of the K+ current are similar to those of thel-type K+ channel current in mature T lymphocytes. The K+ current in FDC-P2 cells was dramatically reduced after culture in the IL-3-free medium for 1–2 days. When IL-3 was re-added to the medium, the current was re-expressed. These observations suggest that expression of the K+ current depends on extracellular IL-3, and that the current may play some roles in proliferation of these cells. 相似文献
2.
Summary The roles that Ca2+, calmodulin, and ATP play in the redistribution of conconavalin A (Con A) binding sites on the surface of mouse T-lymphoma cells were examined. Membranes of cells labeled with fluorescein-conjugated Con A (Fl-Con A) were made permeable (skinned) to ions and proteins by incubation in a solution containing no added Ca2+, 7mm EGTA, and ATP. The intracellular ionic and protein concentrations could then be varied, and the degree of Con A receptor capping monitored simultaneously. A graded increase (9.0 to 30%) was found in the number of capped cells with increasing Ca2+ concentration from 10–6–10–4.9
m. Increasing concentrations of trifluoperazine, chlorpromazine, and promethazine (1.5×10–6 to 1.0×10–4
m) in cell suspensions containing 10–4
m Ca2+ produced graded inhibition of capping in the same order that the drugs bind to calmodulin. Removal of extracellular Ca2+ dissociated (reversed) some of the caps into patches, thus reducing their number (12%). ATP was required for either capping or cap dissociation to occur. Addition of calmodulin (3.9×10–8–6.3×10–7
m) to the cell suspension increased the Ca2+ sensitivity. These results provide direct evidence that capping of Con A receptors is a reversible process (i) regulated by intracellular Ca2+ concentration, (ii) requiring ATP as an energy source, and (iii) susceptible to the influence of calmodulin. These findings are consistent with the hypothesis that the collection of surface receptor patches into cap structures is controlled by the interaction of actomyosin filaments, which in turn is regulated by a Ca2+-calmodulin-activated control system. 相似文献
3.
A. A. Bernardo F. T. Kear O. S. Ruiz J. A. L. Arruda 《The Journal of membrane biology》1994,140(1):31-37
The renal basolateral Na+/HCO
3
–
cotransporter is the main system responsible for HCO
3
–
transport from proximal tubule cells into the blood. The present study was aimed at purifying and functionally reconstituting the Na+/HCO
3
–
cotransporter protein from rabbit renal cortex. Highly purified rabbit renal cortical basolateral membrane vesicles (hereafter designated as original basolateral membrane), enriched 12-fold in Na-K-ATPase, were solubilized in 2% octylglucoside, and then reconstituted in l--phosphatidylcholine (proteoliposomes). Na+/HCO
3
–
cotransporter activity was assessed as the difference in 22Na uptake in the presence of HCO
3
–
and gluconate. The activity of the Na+/HCO
3
–
cotransporter was enhanced 18-fold in the solubilized protein reconstituted into proteoliposomes compared to the original basolateral membranes. The reconstituted solubilized purified protein exhibited kinetic properties similar to the cotransporter from original basolateral membranes. In addition, it was like the original cotransporter, inhibited by disulfonic stilbene SITS, and was eleetrogenic. The catalytic subunit of protein kinase A significantly inhibited Na+/HCO
3
–
cotransporter activity in proteoliposomes. The octylglucoside-solubilized protein was further purified by hydroxylapatite column chromatography, and this resulted in an additional enhancement of Na+/HCO
3
–
cotransporter activity of 80-fold over the original basolateral membranes. The fractions containing the highest activity were further processed by glycerol gradient centrifugation, resulting in a 124- to 300-fold increase in Na+/HCO
3
–
cotransporter activity compared to the original basolateral membranes. SDS-PAGE analysis showed an enhancement of a protein doublet of 56 kD MW in the glycerol gradient fraction. Our results demonstrate that we have partially purified and reconstituted the renal Na+/HCO
3
–
cotransporter and suggest that the 56 kD doublet protein may represent the Na+/HCO
3
–
cotransporter.This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.). 相似文献
4.
Summary The relationship between the external Ca2+ concentrations [Ca2+]0 and the electrical tolerance (breakdown) in theChara plasmalemma was investigated. When the membrane potential was negative beyond –350–400 mV (breakdown potential, BP), a marked inward current was observed, which corresponds to the so-called punch-through (H.G.L. Coster,Biophys. J.
5:669–686, 1965). The electrical tolerance of theChara plasmalemma depended highly on [Ca2+]0. Increasing [Ca2+]0 caused a more negative and decreasing it caused a more positive shift of BP. BP was at about –700 mV in 200 M La3+ solution. [Mg2+]0 depressed the membrane electrical tolerance which was supposed to be due to competition with Ca2+ at the Ca2+ binding site of the membrane. Such a depressive effect of Mg2+ was almost masked when the [Ca2+]0/[Mg2+]0 ratio was roughly beyond 2. 相似文献
5.
Dario Cremaschi Giuliano Meyer Carlo Rossetti Guido Bottà Paola Palestini 《The Journal of membrane biology》1987,95(3):209-218
Summary Cl– influx at the luminal border of the epithelium of rabbit gallbladder was measured by 45-sec exposures to36Cl– and3H-sucrose (as extracellular marker). Its paracellular component was evaluated by the use of 25mm SCN– which immediately and completely inhibits Cl– entry into the cell. Cellular influx was equal to 16.7eq cm–2 hr–1 and decreased to 8.5eq cm–2 hr–1 upon removal of HCO
3
–
from the bathing media and by bubbling 100% O2 for 45 min. When HCO
3
–
was present, cellular influx was again about halved by the action of 10–4
m acetazolamide, 10–5 to 10–4
m furosemide, 10–5 to 10–4
m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS), 10–3
m amiloride. The effects of furosemide and SITS were tested at different concentrations of the inhibitor and with different exposure times: they were maximal at the concentrations reported above and nonadditive. In turn, the effects of amiloride and SITS were not additive. Acetazolamide reached its maximal action after an exposure of about 2 min. When exogenous HCO
3
–
was absent, the residual cellular influx was insensitive to acetazolamide, furosemide and SITS. When exogenous HCO
3
–
was present in the salines, Na+ removal from the mucosal side caused a slow decline of cellular Cl– influx; conversely, it immediately abolished cellular Cl– influx in the absence of HCO
3
–
. In conclusion, about 50% of cellular influx is sensitive to HCO
3
–
, inhibitable by SCN–, acetazolamide, furosemide, SITS and amiloride and furthermore slowly dependent on Na+. The residual cellular influx is insensitive to bicarbonate, inhibitable by SCN–, resistant to acetazolamide, furosemide, SITS and amiloride, and immediately dependent on Na+. Thus, about 50% of apical membrane NaCl influx appears to result from a Na+/H+ and Cl–/HCO
3
–
exchange, whereas the residual influx seems to be due to Na+–Cl– contranport on a single carrier. Whether both components are simultaneously present or the latter represents a cellular homeostatic counterreaction to the inhibition of the former is not clear. 相似文献
6.
Summary The presence of a coupled Na+/Ca2+ exchange system has been demonstrated in plasma membrane vesicles from rat pancreatic acinar cells. Na+/Ca2+ exchange was investigated by measuring45Ca2+ uptake and45Ca2+ efflux in the presence of sodium gradients and at different electrical potential differences across the membrane (=) in the presence of sodium. Plasma membranes were prepared by a MgCl2 precipitation method and characterized by marker enzyme distribution. When compared to the total homogenate, the typical marker for the plasma membrane, (Na++K+)-ATPase was enriched by 23-fold. Markers for the endoplasmic reticulum, such as RNA and NADPH cytochromec reductase, as well as for mitochondria, the cytochromec oxidase, were reduced by twofold, threefold and 10-fold, respectively. For the Na+/Ca2+ countertransport system, the Ca2+ uptake after 1 min of incubation was half-maximal at 0.62 mol/liter Ca2+ and at 20 mmol/liter Na+ concentration and maximal at 10 mol/liter Ca2+ and 150 mmol/liter Na+ concentration, respecitively. When Na+ was replaced by Li+, maximal Ca2+ uptake was 75% as compared to that in the presence of Na+. Amiloride (10–3 mol/liter) at 200 mmol/liter Na+ did not inhibit Na+/Ca2+ countertransport, whereas at low Na+ concentration (25 mmol/liter) amiloride exhibited dose-dependent inhibition to be 62% at 10–2 mol/liter. CFCCP (10–5 mol/liter) did not influence Na+/Ca2+ countertransport. Monensin inhibited dose dependently; at a concentration of 5×10–6 mol/liter inhibition was 80%. A SCN– or K+ diffusion potential (=), being positive at the vesicle inside, stimulated calcium uptake in the presence of sodium suggesting that Na+/Ca2+ countertransport operates electrogenically, i.e. with a stoichiometry higher than 2 Na+ for 1 Ca2+. In the absence of Na+, did not promote Ca2+ uptake. We conclude that in addition to ATP-dependent Ca2+ outward transport as characterized previously (E. Bayerdörffer, L. Eckhardt, W. Haase & 1. Schulz, 1985,J. Membrane Biol.
84:45–60) the Na+/Ca2+ countertransport system, as characterized in this study, represents a second transport system for the extrusion of calcium from the cell. Furthermore, the high affinity for calcium suggests that this system might participate in the regulation of the cytosolic free Ca2+ level. 相似文献
7.
Background
Respiratory syncytial virus (RSV) is the leading cause of respiratory infections in children, elderly, and immunocompromised individuals. Severe infection is associated with short- and long-term morbidity including pneumonia, recurrent wheezing, and abnormal pulmonary function, and several lines of evidence indicate that impaired adaptive immune responses during infection are critical in the pathophysiology of RSV-mediated disease. Myeloid Dendritic cells (mDCs) play a pivotal role in shaping antiviral immune responses in the respiratory tract; however, few studies have examined the interactions between RSV and individual mDC subsets. In this study, we examined the effect of RSV on the functional response of primary mDC subsets (BDCA-1+ and BDCA-3+) isolated from peripheral blood.Methods
BDCA-1+ and BDCA-3+ mDCs were isolated from the peripheral blood of healthy adults using FACS sorting. Donor-matched BDCA-1+ and BDCA-3+ mDCs were infected with RSV at a multiplicity of infection (MOI) of 5 for 40 hours. After infection, cells were analyzed for the expression of costimulatory molecules (CD86, CD80, and PD-L1), cytokine production, and the ability to stimulate allogenic CD4+ T cell proliferation.Results
Both BDCA-1+ and BDCA-3+ mDCs were susceptible to infection with RSV and demonstrated enhanced expression of CD86, and the inhibitory costimulatory molecules CD80 and PD-L1. Compared to BDCA-3+ mDCs, RSV-infected BDCA-1+ mDC produced a profile of cytokines and chemokines predominantly associated with pro-inflammatory responses (IL-1β, IL-6, IL-12, MIP-1α, and TNF-α), and both BDCA-1+ and BDCA-3+ mDCs were found to produce IL-10. Compared to uninfected mDCs, RSV-infected BDCA-1+ and BDCA-3+ mDCs demonstrated a reduced capacity to stimulate T cell proliferation.Conclusions
RSV infection induces a distinct pattern of costimulatory molecule expression and cytokine production by BDCA-1+ and BDCA-3+ mDCs, and impairs their ability to stimulate T cell proliferation.The differential expression of CD86 and pro-inflammatory cytokines by highly purified mDC subsets in response to RSV provides further evidence that BDCA-1+ and BDCA-3+ mDCs have distinct roles in coordinating the host immune response during RSV infection. Findings of differential expression of PD-L1 and IL-10 by infected mDCs, suggests possible mechanisms by which RSV is able to impair adaptive immune responses. 相似文献8.
Macroscopic instantaneous and time-dependent currents have been measured in the vacuolar membrane of Beta vulgaris using a patch clamp configuration analogous to whole cell mode. At low cytosolic Ca2+ and in the absence of Mg2+, only an instantaneous current was observed. This current is carried predominantly by cations (PKPCl 71, pnapcl 41 and arginine is also conducted). The instantaneous current can be activated by ATP4– (e.g., ATP-activated mean K+ current density was –20 mA.m–2 at a membrane voltage of –20 mV) and by increasing cytosolic pH and Mg2+ (raising Mg2+ from 0 to 0.4 mm induced a mean current density increase of –7 mA.m–2 at –20 mV). Such current can be activated by simultaneous addition of putative in vivo concentrations of ATP4–/MgATP/Mg
free
2+
(in the presence of bafilomycin to inhibit the vacuolar ATPase) and further modulated by cytosolic pH. With vacuolar K+ concentration greater than that of the cytosol, activation of the instantaneous current would mediate vacuolar K+ release over the range of physiological membrane voltage. It is argued that the ATP4–-activated current, in addition to acting as a K+ mobilization pathway, could provide a counter-ion (shunt) conductance, allowing the two electrogenic H+ pumps which reside in the vacuolar membrane to acidify the vacuolar lumen.A separate time-dependent current, which was not observed at low Ca2+ concentrations (less than 500 nm) could also be elicited by addition of Mg2+ at the cytoplasmic membrane face. This current was stimulated by increasing cytoplasmic pH.The authors are grateful to the BBSRC for financial support (Grant PG87/529) and to the Royal Society (University Research Fellowship to J.M.D.). We thank C. Abbott, K. Partridge and J. Robinson for plant cultivation; A. Amtmann, A. Bertl, D. Gradmann and G. Thiel for helpful discussion. 相似文献
9.
Patients with head and neck squamous cell carcinoma (HNSCC) have profound immune defects. These defects are associated with a poor prognosis and are mediated, in part, by immune inhibitory CD34+ progenitor cells, whose numbers are increased in the peripheral blood of HNSCC patients. Immune inhibitory CD34+ cells are also present within HNSCC tumors. A phase IB clinical trial was conducted with HNSCC patients to determine if treatment with the differentiation-inducer 25-hydroxyvitamin D3 could diminish CD34+ cell levels and improve a panel of immune parameters. Here we present the results of treatment with orally administered escalating doses (20, 40, 60 g) of 25-hydroxyvitamin D3, with an emphasis on the six patients who received the maximum dosage of 60 g per day. Peripheral blood was collected at 0, 1, 2, 4, and 6 weeks, and assessed for markers of immune activity. Although no clinical responses were observed, results of this pilot study demonstrated that treatment of HNSCC patients with 25-hydroxyvitamin D3 reduces the number of immune suppressive CD34+ cells, increases HLA-DR expression, increases plasma IL-12 and IFN- levels, and improves T-cell blastogenesis. In contrast, 25-hydroxyvitamin D3 treatment did not modulate plasma IL-1, IL-2, IL-4, IL-6, IL-10, GM-CSF, or TGF- levels.Abbreviations GM-CSF
granulocyte-macrophage colony-stimulating factor
- high CD34+ patients
patients with greater than 1% baseline CD34+ cell levels
- HLA
human leukocyte antigen
- IFN
interferon
- IL
interleukin
- low CD34+ patients
patients with less than 1% baseline CD34+ cell levels
- OD
optical density
- TGF
transforming growth factor 相似文献
10.
G. Malnic A. G. Lopes A. C. Cassola A. L. Berardi M. Mello Aires G. Giebisch 《The Journal of membrane biology》1990,118(2):121-130
Summary The pH-stat technique has been used to measure H+ fluxes in gastric mucosa and urinary bladder in vitro while keeping mucosal pH constant. We now report application of this method in renal tubules. We perfused proximal tubules with double-barreled micropipettes, blocked luminal fluid columns with oil and used a double-barreled Sb/reference microelectrode to measure pH, and Sb or 1n HC1-filled microelectrodes to inject OH– or H+ ions into the tubule lumen. By varying current injection, pH was kept constant at adjustable levels by an electronic clamping circuit. We could thus obtain ratios of current (nA) to pH change (apparent H+-ion conductance). These ratios were reduced after luminal 10–4
m acetazolamide, during injection of OH–, but they increased during injection of H+. The point-like injection source causes pH to fall off with distance from the injecting electrode tip even in oil-blocked segments. Therefore, a method analogous to cable analysis was used to obtain H+ fluxes per cm2 epithelium. The relation betweenJ
H
+
and pH gradient showed saturation kinetics of H fluxes, both during OH– and H+ injection. This kinetic behavior is compatible with inhibition ofJ
H by luminal H+. It is also compatible with dependence on Na+ and H+ gradients of a saturable Na/H exchanger. H+-ion back-flux into the tubule lumen also showed saturation kinetics. This suggests that H+ flow is mediated by a membrane component, most likely the Na+–H+ exchanger. 相似文献
11.
Two-month old Wistar rats of both sexes received, as sole drinking liquid, an aqueous solution of ammonium metavanadate (AMV) at a concentration of 0.01 or 0.05 mg V cm–3 (0.2 or 1.0 mM) for a period of 4 weeks. It was calculated that the animals took up doses of 1.5 and 5–6 mg V kg body weight–1 24 h–1, respectively. Food and AMV solution consumption in the experimental groups was similar to food and water consumption in the control group. A statistically significant decrease of consumption of AMV solution at a concentration of 0.05 mg V cm–3 was noted only in males. Hematological examination demonstrated a decrease in the erythrocyte count, hemoglobin level and hematocrit index. This decrease in the erythrocyte count was associated with an increased percentage of reticulocytes in the peripheral blood of the animals drinking the solution with a higher vanadium content. Biochemical analyses demonstrated a decrease of l-ascorbic acid levels in the plasma and erythrocytes of animals drinking the AMV solutions. A distinct tendency for the malonyldialdehyde level to increase in the blood was also observed. Among the enzymes examined in the erythrocytes (catalase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase and -aminolevulinic acid dehydratase [ALA-D]) only ALA-D activity was depressed. 相似文献
12.
Summary K+, Cl–, and Ca2+ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K+, K+ channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K+ channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K+ channels showed a significant discrimination against Na+ and Cl–. The Cl– channel opened at positive vacuolar membrane potentials for cytoplasmic Cl– influx had a high conductance of 110pS in symmetrical 100mM Cl–. When K+ and Cl– channels were excluded from opening, no traces were found of Ca2+ channel activity for vacuolar Ca2+ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca2+ channel which allowed influx of cytoplasmic Ca2+ into the vacuole when the Ca2+ concentration on the cytoplasmic side was high. When Ca2+ was substituted by Ba2+, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba2+Sr2+Ca2+Mg2+=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca2+ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd2+, but was insensitive to La3+, Gd3+, Ni2+, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K+, Cl–, and Ca2+ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells. 相似文献
13.
Giuliano Meyer Guido Bottà Carlo Rossetti Dario Cremaschi 《The Journal of membrane biology》1990,118(2):107-120
Summary In rabbit gallbladder epithelium, a Na+/H+, Cl–/HCO
3
–
double exchange and a Na+–Cl– symport are both present, but experiments on intact tissue cannot resolve whether the two transport systems operate simultaneously. Thus, isolated apical plasma membrane vesicles were prepared. After preloading with Na+, injection into a sodium-free medium caused a stable intravesicular acidification (monitored with the acridine orange fluorescence quenching method) that was reversed by Na+ addition to the external solution. Although to a lesser extent, acidification took place also in experiments with an electric potential difference (PD) equal to 0. If a preset pH difference (pH) was imposed ([H+]in>[H+]out, PD=0), the addition of Na-gluconate to the external solution caused pH dissipation at a rate that followed saturation kinetics. Amiloride (10–4
m) reduced the pH dissipation rate. Taken together, these data indicate the presence of Na+ and H+ conductances in addition to an amiloride-sensitive, electroneutral Na+/H+ exchange.An inwardly directed [Cl–] gradient (PD=0) did not induce intravesicular acidification. Therefore, in this preparation, there was no evidence for the presence of a Cl–/OH– exchange.When both [Na+] and [Cl–] gradients (outwardly directed, PD=0) were present, fluorescence quenching reached a maximum 20–30 sec after vesicle injection and then quickly decreased. The decrease was not observed in the presence of a [Na+] gradient alone or the same [Na+] gradient with Cl– at equal concentrations at both sides. Similarly, the decrease was abolished in the presence of both Na+ and Cl– concentration gradients and hydrochlorothiazide (5×10–4
m). The decrease was not influenced by an inhibitor of Cl–/OH– exchange (10–4
m furosemide) or of Na+–K+–2Cl– symport (10–5
m bumetanide).We conclude that a Na+/H+ exchange and a Na+–Cl– symport are present and act simultaneously. This suggests that in intact tissue the Na+–Cl– symport is also likely to work in parallel with the Na+/H+ exchange and does not represent an induced homeostatic reaction of the epithelium when Na+/H+ exchange is inhibited. 相似文献
14.
Summary The Na+ requirement for active, electrogenic Cl– absorption byAmphiuma small intestine was studied by tracer techniques and double-barreled Cl–-sensitive microelectrodes. Addition of Cl– to a Cl–-free medium bathingin vitro intestinal segments produced a saturable (K
m
=5.4mm) increase in shortcircuit current (I
sc) which was inhibitable by 1mm SITS. The selectivity sequence for the anion-evoked current was Cl–=Br–>SCN–>NO
3
–
>F–=I–. Current evoked by Cl– reached a maximum with increasing medium Na concentration (K
m
=12.4mm). Addition of Na+, as Na gluconate (10mm), to mucosal and serosal Na+-free media stimulated the Cl– current and simultaneously increased the absorptive Cl– flux (J
ms
Cl
) and net flux (J
net
Cl
) without changing the secretory Cl– flux (J
sm
Cl
). Addition of Na+ only to the serosal fluid stimulatedJ
ms
Cl
much more than Na+ addition only to the mucosal fluid in paired tissues. Serosal DIDS (1mm) blocked the stimulation. Serosal 10mm Tris gluconate or choline gluconate failed to stimulateJ
ms
Cl
. Intracellular Cl– activity (a
Cl
i
) in villus epithelial cells was above electrochemical equilibrium indicating active Cl– uptake. Ouabain (1mm) eliminated Cl– accumulation and reduced the mucosal membrane potential
m
over 2 to 3 hr. In contrast, SITS had no effect on Cl– accumulation and hyperpolarized the mucosal membrane. Replacement of serosal Na+ with choline eliminated Cl– accumulation while replacement of mucosal Na+ had no effect. In conclusion by two independent methods active electrogenic Cl– absorption depends on serosal rather than mucosal Na+. It is concluded that Cl– enters the cell via a primary (rheogenic) transport mechanism. At the serosal membrane the Na+ gradient most likely energizes H+ export and regulates mucosal Cl– accumulation perhaps by influencing cell pH or HCO
3
–
concentration. 相似文献
15.
B. N. Ivanov V. L. Shmeleva V. I. Ovchinnikova 《Journal of bioenergetics and biomembranes》1985,17(4):239-249
The number of protons released inside the chloroplast thylakoids per electron which is transferred through the electron transport chain (H+/e– ratio) was measured in isolated pea chloroplasts at pH 6.0 under continuous illumination and with methyl viologen as an electron acceptor. At saturating light intensity (200 W · m–2) (strong light) the H+/e– ratio was 3. At low intensity (0.9 W · m–2) (weak light) the H+/e– ratio was 2 with dark-adapted chloroplasts, but it was close to 3 with chloroplasts that were preilluminated with strong light. It is shown that the presence of azide in the reaction mixture leads to errors in the determination of the H+/e– ratio due to underestimation of the initial rate of H+ efflux on switching off the light. To explain the above data, we assume that transformation of the electron transport chain occurs during illumination with strong light, namely, the Q cycle becomes operative. 相似文献
16.
M. J. Beilby 《The Journal of membrane biology》1981,62(3):207-218
Summary The changes in the cytoplasmic Cl– concentration, [Cl–]
c
, are monitored at the time of withdrawal (starvation) and subsequent replacement of Cl– in the outside medium. The measurement technique exploits the involvement of Cl– inChara excitation. The transient clamp current due to Cl–,I
Cl, is separated from other excitation transients through Hodgkin-Huxley (HH) equations, which have been adjusted toChara. TheI
Cl amplitude depends on HH parameters, [Cl–]
c
and the maximum membrane conductance to Cl–,
. The results are discussed in terms of these quantities.I
Cl and
were found to fall after 6–10 hr of Cl– starvation, thus supporting the hypothesis that [Cl–
c
decreases in Cl–-free medium. The best HH fit to starved data was obtained with [Cl–
c
=3.5mm. The time-course forI
Cl decline is considerably slower than the time-course of the rise of the starvation-stimulated influx. As cells starved for periods longer than 24 hr are re-exposed to Cl–, it is revealed that while [Cl–]
c
remains low during long starvation,
increases to values greater than those of the normal cells. Such differences among cells starved for various lengths of time have not been detected previously. 相似文献
17.
Ian Henry Lambert Else Kay Hoffmann Finn Jørgensen 《The Journal of membrane biology》1989,111(2):113-131
Summary The fluorescence intensity of the dye 1,1-dipropyloxadicarbocyanine (DiOC3-(5)) has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (V
m
) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na+-free K+/choline+ media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155mm. Calibration by the valinomycin null point procedure described by Lariset al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976,Biochim. Biophys. Acta
436:475–488) is shown to be valid only in the presence of the Cl–-channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN– as an indirect estimation of the membrane potential is found not to be applicable for the fast changes inV
m
reported in this paper. Incubation with DiOC3-(5) for 5 min is demenstrated to reduce the Cl– permeability by 26±5% and the NO
3
–
permeability by 15±2%, while no significant effect of the probe could be demonstrated on the K+ permeability. Values forV
m
, corrected for the inhibitory effect of the dye on the anion conductance, are estimated at –61±1 mV in isotonic standard NaCl medium, –78±3 mV in isotonic Na+-free choline medium and –46±1 mV in isotonic NaNO3 medium. The cell membrane is depolarized by addition of the K+ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na+-free choline medium, indicating thatV
m
is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO
3
–
for all cellular and extracellular Cl– leads to a depolarization of the membrane, demonstrating thatV
m
is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K+ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 S/cm2 for K+, 3.0 S/cm2 for Na+, 0.6 S/cm2 for Cl– and 8.7 S/cm2 for NO
3
–
. Addition of the Ca2+-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K+ permeability exceeds the Cl– permeability also after the addition of A23187. The K+ and Cl– conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 S/cm2, respectively. The membrane potential is depolarized in hypotonically swollen cells, confirming that the increase in the Cl– permeability following hypotonic exposure exceeds the concommitant increase in the K+ permeability. In control experiments where the membrane potentialV
m
=E
K
=E
Cl
=E
Na
, it is demonstrated that cell volume changes has no significant effect on the fluorescence signal, apparently because of a large intracellular buffering capacity. The increase in the Cl– conductances is 68-fold when cells are transferred to a medium with half the osmolarity of the standard medium, as estimated from the net Cl– efflux and the change inV
m
. The concommitant increase in the K+ conductance, as estimated from the net K+ efflux, is only twofold. 相似文献
18.
Cl− channels in basolateral renal medullary memnbranes: III. Determinants of single-channel activity
Christopher J. Winters W. Brian Reeves Thomas E. Andreoli 《The Journal of membrane biology》1990,118(3):269-278
Summary We evaluated the effects of vawrying aqueous Cl– concentrations, and of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO), on the properties of Cl– channels fused from basolaterally enriched renal medullary vesicles into planar lipid bilayers. The major channel properties studied were the anion selectivity sequence, anionic requirements for, channel activity. and the efects of varying Cl– concentrations and/or PGO on the relation between holding voltageV
H
-mV) and open-time probability (P
o).Reducingcis Cl– concentrations, in the range 50–320mm, produced a linear reduction in fractional open time (P
v) with a half-maximal reduction inP
o atcis Cl–170mM. Channel activity was sustained by equimolar replacement ofcis Cl– with F–, but not with impermeant isethionate. Fortrans solutions, the relation between Cl– concentration andP
0 at 10mm Cl–. Reducingcis Cl– had no effect on the gating charge (Z) for channel opening, but altered significantly the voltage-independent, energy (G) for channel opening.Phenylglyoxal (PGO) reducedZ and altered G for Cl– channel activity when added tocis, but nottrans solutions, Furthermore, in the presence ofcis PGO, reducing thecis Cl– concentration had no effect onZ but altered G. Thus we propose thatcis PGO and,cis Cl– concentrations affect separate sites determining channel activity at the extracellular faces of, these Cl– channels. 相似文献
19.
Ilse Foissner 《Protoplasma》1990,154(2-3):80-90
Summary The formation of wall appositions (plugs) by ionophore A 23187, CaCl2, LaCl3, and nifedipine was studied in mature internodal cells of characeaen algae. CaCl2 at concentrations above 10–2M induces thick fibrillar plugs without callose inNitella flexilis. InChara corallina andNitella flexilis ionophore A 23187 (1.25×10–5 to 5×10–5M) and LaCl3 (7.5×10–5 to 2.5×10–4M) cause flat appositions which contain callose and have a more granular structure. Plug formation by ionophore A 23187, CaCl2, and LaCl3 is pH-dependent and occurs beneath the alkaline regions of the cell. Nifedipine (10–4 to 10–5M) induces plugs inNitella flexilis after previous injury. These callose-containing wall appositions consist of a heterogeneous granular core which is covered by a fibrillar layer. The results of this work are compared with previous studies on wound wall formation and chlortetracycline (CTC)-induced plug formation which reveal that abundant coated vesicles occur only when a thick fibrillar wall layer is formed. Neither LaCl3 nor nifedipine inhibit the formation of CaCl2- or CTC-plugs. The unusual effects of these substances, which normally act as Ca2+ antagonists and therefore should prevent and not induce plug formation, are discussed. It is suggested that La3+ mimicks the effects of calcium and that nifedipine binding to the Ca2+ channels is altered in the alkaline regions of characean internodes and allows an influx of Ca2+.Abbreviations AFW
artificial fresh water
- CTC
chlortetracycline
- DCMU
dichlorphenyldimethylurea
- DMSO
dimethylsulfoxide
- EGTA
ethyleneglycoltetraacetic acid
- MES
2-(N-morpholino) ethanesulfonic acid
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- TAPS
N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid 相似文献
20.
Summary In order to demonstrate the presence of a Ca2+-activated Cl–-channel in theNitellopsis plasmalemma, tonoplast-free cells were prepared and their intracellular Ca2+ concentration was modified by internal perfusion. An increase in the Ca2+ concentration caused a large Cl– efflux with a concomitant depolarization of the membrane potential. These changes were for the most part reversible. The critical Ca2+ concentration was about 4.0 m. Neither the Cl– efflux nor the membrane depolarization showed a time-dependent inactivation. A Cl–-channel blocker, A-9-C (9-anthracenecarboxylic acid) reduced both the Cl– efflux and the magnitude of the membrane potential depolarization. A small increase in the intracellular Ca2+ concentration, which is caused by membrane excitation of tonoplast-free cells is not sufficient to activate this Ca2+-dependent Cl–-channel. 相似文献