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1.
A self-referencing and non-invasive Ca 2+-sensitive vibrating electrode was used to assess the effects of hydrogen peroxide-induced oxidative challenges on the efflux and influx of calcium across the plasma membrane of single nerve cells cultured from abdominal ganglion of Aplysia californica. A reduced net efflux of Ca 2+ from the cell soma occurred immediately after the addition of hydrogen peroxide (0.0025 mM, 0.005 mM or 0.01 mM) to the culture medium, indicating damage to the cell membrane or Ca 2+ transport mechanism. There then followed a marked efflux, the extent and duration of which was related to the concentration of hydrogen peroxide used and which may reflect compensatory activity by the Ca 2+ regulatory mechanisms in the plasmalemma. No morphological changes were observed in cells challenged with 0.0025 mM hydrogen peroxide and the enhanced rate of Ca 2+ efflux rapidly decreased to pre-exposure values. Sustained and enhanced Ca 2+ effluxes from those cells exposed to 0.005 mM or 0.01 mM hydrogen peroxide were also consistent with regulatory pumping of Ca 2+ out of the cell although contraction and blebbing of neurites and swelling of the soma may indicate that a proportion of the efflux arose from release of Ca 2+ from disrupted intracellular stores. The vibrating electrode is a useful additional technique for the study of the pathogenesis of neurological conditions, as ionic fluxes across single nerve cells exposed to physiologically-relevant concentrations of free radicals can be monitored non-invasively for prolonged periods. 相似文献
2.
Abstract Using the method of compartmental analysis, the ion fluxes and compartment concentrations of Ca 2+, K + and Cl - have been compared in the untreated vegetative frond and the abscisic acid (ABA) induced turion of Spirodela polyrrhiza. The ABA-induced turion is characterized by reduced Ca 2+ exchange across the tonoplast and low vacuolar Ca 2+ concentration relative to the vegetative frond. In addition the turion exhibits a higher plasmalemma flux with a correspondingly high Ca 2+ concentration in the cytoplasm. The concentration of K + and Cl - is much lower in the cytoplasm of the ABA-induced turion than in the vegetative frond with the influx/efflux ratio at both the plasmalemma and the tonoplast being less than 1, a finding exhibited also in dormant storage tissue. Treatment of vegetative fronds with ABA for 18 h resulted in a reduced K + plasmalemma efflux relative to untreated vegetative fronds and a concomitant increase in the cytoplasmic concentration. There was no rapid effect of ABA on Ca 2+, K + or Cl - fluxes through either membrane. These results are consistent with the notion that drastic changes in ion fluxes and concentrations in the turion are a secondary consequence of ABA-induced development, possibly due to prior regulation by ABA of enzymes inherent to processes involved in membrane transport. 相似文献
3.
Artificial pH gradients across tonoplast vesicles isolated from storage tissue of red beet ( Beta vulgaris L.) were used to study the kinetics of a Ca 2+/H + antiport across this membrane. Ca 2+-dependent H + fluxes were measured by the pH-dependent fluorescence quenching of acridine orange. ΔpH-dependent Ca 2+ influx was measured radiometrically. Both H + efflux and Ca 2+ influx displayed saturation kinetics and an identical dependence on external calcium with apparent Km values of 43.9 and 41.7 micromolar, respectively. Calcium influx was unaffected by an excess of Mg 2+ but was inhibited by La 3+ > Mn 2+ > Cd 2+. The apparent Km for external calcium was greatly affected (5-fold) by internal pH in the range of 6.0 to 6.5 and a transmembrane effect of internal proton binding on the affinity for external calcium is suggested. 相似文献
4.
Intracellular Ca 2+ level is under strict regulation through calcium channels and storage pools including the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which cause mis-regulated Ca 2+ influx, induce the excitotoxic necrosis of neurons. In the nematode Caenorhabditis elegans, dominant mutations in the DEG/ENaC sodium channel subunit MEC-4 induce six mechanosensory (touch) neurons to undergo excitotoxic necrosis. These necrotic neurons are subsequently engulfed and digested by neighboring hypodermal cells. We previously reported that necrotic touch neurons actively expose phosphatidylserine (PS), an “eat-me” signal, to attract engulfing cells. However, the upstream signal that triggers PS externalization remained elusive. Here we report that a robust and transient increase of cytoplasmic Ca 2+ level occurs prior to the exposure of PS on necrotic touch neurons. Inhibiting the release of Ca 2+ from the ER, either pharmacologically or genetically, specifically impairs PS exposure on necrotic but not apoptotic cells. On the contrary, inhibiting the reuptake of cytoplasmic Ca 2+ into the ER induces ectopic necrosis and PS exposure. Remarkably, PS exposure occurs independently of other necrosis events. Furthermore, unlike in mutants of DEG/ENaC channels, in dominant mutants of deg-3 and trp-4, which encode Ca 2+ channels, PS exposure on necrotic neurons does not rely on the ER Ca 2+ pool. Our findings indicate that high levels of cytoplasmic Ca 2+ are necessary and sufficient for PS exposure. They further reveal two Ca 2+-dependent, necrosis-specific pathways that promote PS exposure, a “two-step” pathway initiated by a modest influx of Ca 2+ and further boosted by the release of Ca 2+ from the ER, and another, ER-independent, pathway. Moreover, we found that ANOH-1, the worm homolog of mammalian phospholipid scramblase TMEM16F, is necessary for efficient PS exposure in thapsgargin-treated worms and trp-4 mutants, like in mec-4 mutants. We propose that both the ER-mediated and ER-independent Ca 2+ pathways promote PS externalization through activating ANOH-1. 相似文献
5.
Measurements were made of the influx of 45Ca into internodal cells of Chara corallina in solutions containing high concentrations of NaCl. Increasing salinity in the range 4–100mol m ?3 NaCl resulted in a doubling of Ca 2+ influx at the plasmalemma. A time-course of Ca 2+ influx in 50 mol m ?3 NaCl, 0.5mol m ?3 CaCl 2 showed that while influx at the plasmalemma increased only 1.5-fold, influx to the vacuole increased by up to 15-fold. This was interpreted as being due to inhibition of active Ca 2+ efflux from the cell. The stimulation of Ca 2+ influx by increasing salinity appeared to be principally a response to reduced turgor since similar stimulations were obtained when turgor was reduced by NaCl, Na 2SO 4 or mannitol. When cells were plasmolysed Ca 2+ influx increased by 10–20-fold. The increased permeability was relatively specific for Ca 2+ and was inhibitable by La 3+. Survival of cells in high salt conditions was increased by 30 mmol m ?3 La 3+, which inhibited Ca 2+ influx. Paradoxically, survival can also be extended by increasing external Ca 2+ which leads to a higher influx. Therefore, it seems unlikely that the ameliorative effect of Ca 2+ on the sensitivity of plants to high NaCl is mediated by Ca 2+ entry across the plasmalemma. It seems more likely that the principal role of Ca 2+ under these conditions is exerted externally through the control of membrane voltage and permeability. 相似文献
6.
The characteristics of Ca 2+ transport across the excitable membrane of Paramecium aurelia were studied by measuring 45Ca 2+ influx and efflux. The intracellular concentration of free Ca 2+ in resting P. aurelia was at least ten times less than the extracellular concentration. Ca 2+ influx was easily measurable at 0°C, but not at 23°C. The influx of 45Ca 2+ was stimulated by the same conditions which cause membrane depolarization and ciliary reversal. Addition of Na + and K + (which stimulate ciliary reversal) resulted in a 10-fold increase in the rate of Ca 2+ influx. An externally applied, pulsed, electric field (1–2 mA/cm 2 of electrode surface), caused the rate of Ca 2+ influx to increase 3–5 times, with the extent of stimulation dependent on the current density and the pulse width Ca 2+ influx had the characteristics of a passive transport system and was associated with the chemically or electrically triggered Ca 2+ “gating” mechanism, which has been studied electrophysiologically. In contrast, Ca 2+ efflux appeared to be catalyzed by an active transport system. With cells previously loaded at 0°C with 45Ca 2+, Ca 2+ efflux was rapid at 23°C, but did not occur at 0°C. This active Ca 2+ efflux mechanism is probably responsible for maintaining the low internal Ca 2+ levels in unstimulated cells. 相似文献
7.
Abstract: The features of Ca 2+ fluxes, the importance of the Ca 2+ pump‐mediated H +/Ca 2+ exchanges at plasmalemma level, and the possible involvement of Ca 2+‐ATPase activity in ABA‐induced changes of H + fluxes were studied in Egeria densa leaves. The results presented show that, while in basal conditions no net Ca 2+ flux was evident, a conspicuous Ca 2+ influx (about 1.1 ìmol g ?1 FW h ?1) occurred. The concomitant efflux of Ca 2+ was markedly reduced by treatment with 5 íM eosin Y (EY), a specific inhibitor of the Ca 2+‐ATPase, that completely blocked the transport of Ca 2+ after the first 20 ‐ 30 min. The decrease in Ca 2+ efflux induced by EY was associated with a significant increase in net H + extrusion (?ÄH +) and a small but significant cytoplasmic alkalinization. The shift of external [Ca 2+] from 0.3 to 0.2 mM (reducing Ca 2+ uptake by about 30 %) and the hindrance of Ca 2+ influx by La 3+ were accompanied by progressively higher ?ÄH + increases, in agreement with a gradual decrease in the activity of a mechanism counteracting the Ca 2+ influx by an nH +/Ca 2+ exchange. The ABA‐induced decreases in ?ÄH + and pH cyt were accompanied by a significant increase in Ca 2+ efflux, all these effects being almost completely suppressed by EY, in line with the view that the ABA effects on H + fluxes are due to activation of the plasmalemma Ca 2+‐ATPase. These results substantially stress the high sensitivity and efficacy of the plasmalemma Ca 2+ pump in removing from the cytoplasm the Ca 2+ taken up, and the importance of the contribution of Ca 2+ pump‐mediated H +/Ca 2+ fluxes in bringing about global changes of H + fluxes at plasmalemma level. 相似文献
8.
An early event in the hypersensitive response of tobacco to Pseudomonas syringae pv syringae is the initiation of a K +/H + response characterized by specific plasma membrane K + efflux, extracellular alkalinization, and intracellular acidification. We investigated the role of calcium in induction of these host responses. Suspension-cultured tobacco cells exhibited a baseline Ca 2+ influx of 0.02 to 0.06 micromole per gram per hour as determined from 45Ca 2+ uptake. Following bacterial inoculation, uptake rates began to increase coincidently with onset of the K +/H + response. Rates increased steadily for 2 to 3 hours, reaching 0.5 to 1 micromole per gram per hour. This increased Ca 2+ influx was prevented by EGTA and calcium channel blockers such as La 3+, Co 2+, and Cd 2+ but not by verapamil and nifedipine. Lanthanum, cobalt, cadmium, and EGTA inhibited the K +/H + response in both suspension-cultured cells and leaf discs and prevented hypersensitive cell death in leaf discs. We conclude that increased plasmalemma Ca 2+ influx is required for the K +/H + and hypersensitive responses in tobacco. 相似文献
9.
1. A vibrating calcium (Ca 2+)-selective electrode measured Ca 2+ flux across the membrane of trabeculae from the ventricle of the marine gastropod, Busycon canaliculatum. Because the neuropeptide FMRFamide increases both systolic force and rate in the hearts of most mollusc species, the present experiments were conducted to study how Ca 2+ may be mobilized by FMRFamide during excitation-contraction coupling (E-C coupling). 2. Ca 2+ efflux was consistently recorded from the trabeculae in response to FMRFamide. This efflux was the result of the sarcolemma redistributing Ca 2+ into the extracellular compartment after a preceding rapid Ca 2+ influx. Ca 2+ efflux stimulated by FMRFamide was blocked by the l-type Ca 2+ channel blocker verapamil. Conversely, diltiazem potentiated FMRFamide responses. Neither verapamil of diltiazem alone had any effect on spontaneous basal efflux. However, if FMRFamide was present, the membrane was responsive to the action of the Ca 2+ channel blockers, suggesting that a use-dependent mechanism was involved. 3. During spontaneous basal efflux, the Na-Ca exchanger was responsible for only 20% of the total Ca 2+ efflux while during FMRFamide treatment the Na–Ca exchanger may have contributed about 60% to the total Ca 2+ efflux. FMRFamide may not only alter ion channel activity but may also indirectly regulate Ca 2+ extrusion mechanisms during cardioexcitation. 相似文献
10.
Epinephrine stimulated adenylate cyclase in turkey erythrocyte ghosts is inhibited by calcium. The inhibition of adenylate cyclase is not apparent when intact erythrocytes are incubated with calcium and epinephrine. However, in the presence of the specific cation ionophore A 23187 and 5 mm Ca 2+, a 90% inhibition of epinephrine stimulated 3′,5′-adenosine monophosphate formation is found. The effect of catecholamines on calcium transport in the intact turkey erythrocyte was studied. Epinephrine causes a small but significant increase in Ca 2+ efflux. This effect is inhibited by propranolol. No effect of epinephrine on Ca 2+ uptake was observed. However, a 22% increase in Ca 2+ uptake in the presence of propranolol could be detected. The propranolol effect was found to possess high statistical significance ( p < .001). The absence of an epinephrine effect on influx probably reflects the presence of endogenous catecholamines in the control samples.It is proposed that the activation of adenylate cyclase by catecholamines occurs in two phases. The first phase is the increase of net Ca 2+ efflux from a crucial Ca 2+ pool, thus removing Ca 2+ from its inhibitory sites on the adenylate cyclase complex. The second phase is the activation of the deinhibited adenylate cyclase by the hormone. 相似文献
11.
A study of calcium ion regulation in Anabaena 7120 and its derivative mutant (CSE2) strain impaired in ntcA gene were investigated in terms of altered morphological and physiological responses against various levels of calcium stress (0–100 mM). Calcium concentration of 10 mM was found to be inhibitory while 100 mM proved lethal for both wild type and mutant strain. The involvement of Ca 2+ in the regulation of cellular processes has been described in terms of an influx or efflux of Ca 2+ from the cytosol. A biphasic calcium uptake with difference in calcium influx and efflux rate was responsible for differential amount of remaining calcium which followed a decreasing trend both for wild type and mutant. Low K s 0.5 and high V max in mutant suggest heavy and less restricted influx of calcium ion. Further, the interactive effect of calcium influx/efflux rate, remaining Ca 2+ and intracellular levels of Na + and K + may be attributed for the degree of membrane damage and growth sustenance during exogenous supply of calcium salt. Widening in heterocyst spacing pattern, decreased heterocyst frequency and formation of abnormal cell structures at higher concentration (100 mM CaCl 2) suggest that calcium mediated regulatory process modulate heterocyst frequency and maintenance of cell structure. Further, poor regulation of calcium ion homeostasis in ntcA suggests that the calcium level and ntcA gene expression are inter-related. 相似文献
12.
Summary The influence of K + on the Na + fluxes of barley root cells was investigated. A increased K + concentration (K + influx) results in a transient increase of the plasmalemma efflux of Na + followed by a decrease, and in a decrease of the cytoplasmic content and the tonoplast influx of Na +. These results are consistent with a Na-K-pump at the plasmalemma. 相似文献
13.
Washing corn ( Zea mays L.) root tissue in water causes loss of about one-third of the exchangeable Ca 2+ over the first 10 to 15 minutes. Upon transfer to K +-containing solutions, the tissue shows a short period of rapid K + influx which subsequently declines. Addition of 0.1 millimolar Ca 2+ decreases the initial rapid K + influx, but increases the sustained rate of K + and Cl − uptake. It was confirmed (Elzam and Hodges 1967 Plant Physiol 42: 1483-1488) that 0.1 millimolar Ca 2+ is more effective than higher concentrations for the initial inhibition, and that Mg 2+ will substitute. The inhibition arises from a mild shock affect of restoring Ca2+. With 0.1 millimolar Ca2+ net H+ efflux is blocked for 10 to 15 minutes and the cells are depolarized by about 30 millivolts. However, 1 millimolar Ca2+ rapidly produces increased K+ influx and blocks net H+ efflux for only a few minutes; blockage is preceded by a brief net H+ influx which may restore and increase ion transport by reactivating the plasmalemma H+-ATPase. Stimulation of electrogenic H+-pumping with fusicoccin eliminates the shock responses and minimizes Ca2+ effects on K+ influx. Fusicoccin also strongly decreases Ca2+ influx, but has no effect on Ca2+ efflux. Ice temperatures and high pH decreased Ca2+ efflux, but uncoupler and chlorpromazine did not. It is suggested that the inhibitory and promotive actions of Ca2+ are manifested through decreases or increases in the protonmotive force. 相似文献
14.
Opening and closing of the stomatal pore is associated with very large changes in K-salt accumulation in stomatal guard cells. This review discusses the ionic relations of guard cells in relation to the general pattern of transport processes in plant cells, in plasmalemma and tonoplast, involving primary active transport of protons, proton-linked secondary active transport, and a number of gated ion channels. The evidence available suggests that the initiation of stomatal opening is regulated through the uptake mechanisms, whereas initiation of stomatal closing is regulated by control of ion efflux at the plasmalemma, and of fluxes to and from the vacuole. In response to a closing signal there are large transient increases in efflux of both Cl ? (or Br ?) and Rb + (K +) at the plasmalemma, with also a probable increase in anion flux from vacuole to cytoplasm and decrease in anion flux from cytoplasm to vacuole. A speculative hypothetical sequence of events is discussed, by which the primary response to a closing signal is an increase in Ca 2+ influx at the plasmalemma, producing depolarisation and increase in cytoplasmic Ca 2+. The consequent opening of Ca 2+-sensitive Cl ? channels, and voltage-sensitive K + channels (also Ca 2+-sensitive?) in the plasmalemma, and of a Ca 2+-sensitive nonspecific channel in the tonoplast, could produce the flux effects identified by the tracer work; this speculation is also consistent with the Ca 2+-sensitivity of the response to closing signals and with evidence from patch clamping that such channels exist in at least some plant cells, though not yet all shown in guard cells. 相似文献
15.
The flux ratio (influx/efflux) of K + across the plasmalemma of beet cells at an external potassium concentration of 0.6 m m does not respond to changes of membrane potential in the manner expected for the free diffusion of ions. The K + efflux is affected by the presence of adsorbed Ca 2+, but is apparently unrelated to the electrical potential or to the net uptake of potassium. The K + efflux is greater than the efflux of the sulfate and organic anions which are accumulated with potassium, and is partially dependent on the presence of external potassium. Thus the loss of 42K from the cell does not appear to be a leakage of freely diffusing K + ions, nor a leakage of ion pairs, but a carrier-mediated transport or exchange of potassium across the cell membrane. 相似文献
16.
There is evidence for a role of increased cytoplasmic Ca 2+ in the stomatal closure induced by abscisic acid (ABA), but two points of controversy remain the subject of vigorous debate—the universality of Ca 2+ as a component of the signaling chain, and the source of the increased Ca 2+, whether influx across the plasmalemma, or release from internal stores. We have addressed these questions by patch-clamp studies on guard cell protoplasts of Vicia faba, assessing the effects of ABA in the presence and absence of external Ca 2+, and of internal Ca 2+ buffers to control levels of cytoplasmic Ca 2+. We show that ABA-induced reduction of the K + inward rectifier can occur in the absence of external Ca 2+, but is abolished when Ca 2+ buffers are present inside the cell. Thus, some minimum level of cytoplasmic Ca 2+ is a necessary component of the signaling chain by which ABA decreases the K + inward rectifier in stomatal guard cells, thus preventing stomatal opening. Release of Ca 2+ from internal stores is capable of mediating the response, in the absence of any Ca 2+ influx from the extracellular medium. The work also shows that enhancement of the K + outward rectifier by ABA is Ca 2+ independent, and that other signaling mechanisms must be involved. A role for internal pH, as suggested by H.R. Irving, C.A. Gehring and R.W. Parish ( Proc. Natl. Acad. Sci. USA
89:1790–1794, 1990) and M.R. Blatt ( J. Gen. Physiol.
99:615–644, 1992), is an attractive working hypothesis. 相似文献
17.
Internodal cells of a brackish water charophyte, Lamprothamnium succinctum (A. Br. in Ash.) R.D.W. regulate the turgor pressure in response to changes in both the cellular and the external osmotic
pressures. During turgor regulation upon hypotonic treatment, net effluxes of K + and Cl − from the vacuole, membrane depolarization, a transient increase in the electrical membrane conductance and a transient increase
in concentration of cytoplasmic Ca 2+ are induced. Activation of the plasmalemma Ca 2+ channels and the Ca 2+-controlled passive effluxes of K + and Cl − through the plasmalemma ion channels are postulated. 相似文献
18.
The role of Ca 2+ in the initiation and maintenance of contraction has been extensively studies. Many of these studies have focused on how Ca 2+ influx and efflux affect cytoplasmic Ca 2+ (Ca i) and, therefore, contraction in cardiac muscle. However, it has recently become apparent that Ca i itself may play a major role in the control of Ca 2+ influx and efflux from cardiac muscle. Here we review current ideas on the mechanisms underlying Ca 2+ homeostasis in cardiac muscle, with specific attention to how Ca i may control Ca 2+ influx, both under normal and pathological conditions. 相似文献
19.
Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC − toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca 2+ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca 2+ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca 2+ influx promoted by molecules locked in a Ca 2+-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux. 相似文献
20.
Arctigenin, a lignan extract from Arctium lappa (L.), exhibits anti-inflammation, antioxidation, vasodilator effects, etc. However, the effects of arctigenin on bronchus relaxation are not well investigated. This study aimed to investigate how arctigenin regulates bronchus tone and calcium ion (Ca 2+) flow. Trachea strips of guinea pigs were prepared for testing the relaxation effect of arctigenin to acetylcholine, histamine, KCl, and CaCl 2, respectively. Furthermore, l-type calcium channel currents were detected by patch–clamp, and intracellular Ca 2+ concentration was detected by confocal microscopy. The results showed that arctigenin exhibited relaxation effect on tracheae to different constrictors, and this was related to decreasing cytoplasmic Ca 2+ concentration by inhibiting Ca 2+ influx partly through l-type calcium channel as well as promoting Ca 2+ efflux. In summary, this study provides new insight into the mechanisms by which arctigenin exhibits relaxation effect on bronchus and suggests its potential use for airway disease therapy. 相似文献
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