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1.
R.R. Preston 《The Journal of membrane biology》1998,164(1):11-24
The properties of Mg2+ conductances in Paramecium tetraurelia were investigated under two-electrode voltage clamp. When bathed in physiological Mg2+ concentrations (0.5 mm), depolarizing steps from rest elicited a prominent Mg2+-specific current (I
Mg) that has been noted previously. The dependence of this current on extracellular Mg2+ approximated that of Mg2+-induced backward swimming, demonstrating that I
Mg contributes to normal membrane excitation and behavior in this ciliate. Closer analysis revealed that the Mg2+ current deactivated biphasically. While this might suggest the involvement of two Mg2+-specific pathways, both tail-current components were affected similarly by current-specific mutations and they had similar
ion selectivities, suggesting a common pathway. In contrast, a Mg2+ current activated upon hyperpolarization could be separated into three components. The first, I
Mg, had similar properties to the current activated upon depolarization. The second was a nonspecific divalent cation current
(I
NS) that was revealed following suppression of I
Mg by eccentric mutation. The final current was relatively minor and was revealed following suppression of I
Mg and I
NS by obstinate A gene mutation. Reversal-potential analyses suggested that I
Mg and I
NS define two intracellular compartments that contain, respectively, low (0.4 mm) and high (8 mm) concentrations of Mg2+. Measurement of intracellular free Mg2+ using the fluorescent dye, Mag-fura-2, suggested that bulk [Mg2+]
i
rests at around 0.4 mm in Paramecium.
Received: 12 January 1998/Revised: 16 March 1998 相似文献
2.
The concentration of intracellular calcium, [Ca2+]
i
, in Paramecium was imaged during cold-sensitive response by monitoring fluorescence of two calcium-sensitive dyes, Fluo-3 and Fura-Red.
Cooling of a deciliated Paramecium caused a transient increase in [Ca2+]
i
at the anterior region of the cell. Increase in [Ca2+]
i
was not observed at any region in Ca2+-free solution. Under the electrophysiological recording, a transient depolarization of the cell was observed in response
to cooling. On the voltage-clamped cell, cooling induced a transient inward current under conditions where K+ currents were suppressed. These membrane depolarizations and inward currents in response to cooling were lost upon removing
extracellular Ca2+. The cold-induced inward current was lost upon replacing extracellular Ca2+ with equimolar concentration of Co2+, Mg2+ or Mn2+, but it was not affected significantly by replacing with equimolar concentration of Ba2+ or Sr2+. These results indicate that Paramecium cells have Ca2+ channels that are permeable to Ca2+, Ba2+ and Sr2+ in the anterior soma membrane and the channels are opened by cooling.
Received: 1 April 1996/Revised: 23 July 1996 相似文献
3.
K.D. Clark T.M. Hennessey D.L. Nelson R.R. Preston 《The Journal of membrane biology》1997,157(2):159-167
Paramecium tetraurelia responds to extracellular GTP (≥ 10 nm) with repeated episodes of prolonged backward swimming. These backward swimming events cause repulsion from the stimulus
and are the behavioral consequence of an oscillating membrane depolarization. Ion substitution experiments showed that either
Mg2+ or Na+ could support these responses in wild-type cells, with increasing concentrations of either cation increasing the extent of
backward swimming. Applying GTP to cells under voltage clamp elicited oscillating inward currents with a periodicity similar
to that of the membrane-potential and behavioral responses. These currents were also Mg2+- and Na+-dependent, suggesting that GTP acts through Mg2+-specific (I
Mg) and Na+-specific (I
Na) conductances that have been described previously in Paramecium. This suggestion is strengthened by the finding that Mg2+ failed to support normal behavioral or electrophysiological responses to GTP in a mutant that specifically lacks I
Mg (``eccentric'), while Na+ failed to support GTP responses in ``fast-2,' a mutant that specifically lacks I
Na. Both mutants responded normally to GTP if the alternative cation was provided. As I
Mg and I
Na are both Ca2+-dependent currents, the characteristic GTP behavior could result from oscillations in intracellular Ca2+ concentration. Indeed, applying GTP to cells in the absence of either Mg2+ or Na+ revealed a minor inward current with a periodicity similar to that of the depolarizations. This current persisted when known
voltage-dependent Ca2+ currents were blocked pharmacologically or genetically, which implies that it may represent the activation of a novel purinergic-receptor–coupled
Ca2+ conductance.
Received: 28 October 1996/Revised: 24 December 1996 相似文献
4.
A Paramecium cell responded to heat and cold stimuli, exhibiting increased frequency of directional changes in its swimming behavior.
The increase in the frequency of directional changes was maintained during heating, but was transient during cooling. Although
variations were large, as expected with this type of electrophysiological recording, results consistently showed a sustained
depolarization of deciliated cells in response to heating. Depolarizations were also consistently observed upon cooling. However,
these depolarizations were transient and not continuous throughout the cooling period. These depolarizations were lost or
became small in Ca2+-free solutions. In a voltage-clamped cell, heating induced a continuous inward current and cooling induced a transient inward
current under conditions where K+ currents were suppressed. The heat-induced inward current was not affected significantly by replacing extracellular Ca2+ with equimolar concentrations of Ba2+, Sr2+, Mg2+, or Mn2+, and was lost upon replacing with equimolar concentration of Ni2+. On the other hand, the cold-induced inward current was not affected significantly by Ba2+, or Sr2+, however the decay of the inward current was slowed and was lost or became small upon replacing with equimolar concentrations
of Mg2+, Mn2+, or Ni2+. These results indicate that Paramecium cells have heat-activated Ca2+ channels and cold-activated Ca2+ channels and that the cold-activated Ca2+ channel is different from the heat-activated Ca2+ channel in the ion selectivity and the calcium-dependent inactivation.
Received: 9 September 1998/Revised: 22 January 1999 相似文献
5.
The polyamine secretagogue, aminoethyldextran (AED), causes a cortical [Ca2+] transient in Paramecium cells, as analyzed by fluorochrome imaging. Our most essential findings are: (i) Cortical Ca2+ signals also occur when AED is applied in presence of the fast Ca2+ chelator, BAPTA. (ii) Extracellular La3+ application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological
[Ca2+]
i
transient (while injected La3+ causes a sustained fluorescence signal). (iii) Simply increasing [Ca2+]
o
causes a similar rapid, short-lived [Ca2+]
i
transient. All these phenomena, (i–iii), are compatible with activation of an extracellular ``Ca2+/(polyvalent cation)-sensing receptor' known from some higher eukaryotic systems, where this sensor (responding to Ca2+, La3+ and some multiply charged cations) is linked to cortical calcium stores which, thus, are activated. In Paramecium, such subplasmalemmal stores (``alveolar sacs') are physically linked to the cell membrane and they can also be activated
by the Ca2+ releasing agent, 4-chloro-m-cresol, just like in Sarcoplasmic Reticulum. Since this drug causes a cortical Ca2+ signal also in absence of Ca2+
o
we largely exclude a ``Ca2+-induced Ca2+ release' (CICR) mechanism. Our finding of increased cortical Ca2+ signals after store depletion and re-addition of extracellular Ca2+ can be explained by a ``store-operated Ca2+ influx' (SOC), i.e., a Ca2+ influx superimposing store activation. AED stimulation in presence of Mn2+
o
causes fluorescence quenching in Fura-2 loaded cells, indicating involvement of unspecific cation channels. Such channels,
known to occur in Paramecium, share some general characteristics of SOC-type Ca2+ influx channels. In conclusion, we assume the following sequence of events during AED stimulated exocytosis: (i) activation
of an extracellular Ca2+/polyamine-sensing receptor, (ii) release of Ca2+ from subplasmalemmal stores, (iii) and Ca2+ influx via unspecific cation channels. All three steps are required to produce a steep cortical [Ca2+] signal increase to a level required for full exocytosis activation. In addition, we show formation of [Ca2+] microdomains (≤0.5 μm, ≤33 msec) upon stimulation.
Received: 30 August 1999/Revised: 1 December 1999 相似文献
6.
M.-P. Blanchard N. Klauke S. Zitzmann H. Plattner 《The Journal of membrane biology》1999,169(3):155-165
We analyzed [Ca2+]
i
transients in Paramecium cells in response to veratridine for which we had previously established an agonist effect for trichocyst exocytosis (Erxleben
& Plattner, 1994. J. Cell Biol.
127:935–945; Plattner et al., 1994. J. Membrane Biol.
158:197–208). Wild-type cells (7S), nondischarge strain nd9–28°C and trichocyst-free strain ``trichless' (tl), respectively,
displayed similar, though somewhat diverging time course and plateau values of [Ca2+]
i
transients with moderate [Ca2+]
o
in the culture/assay fluid (50 μm or 1 mm). In 7S cells which are representative for a normal reaction, at [Ca2+]
o
= 30 nm (c.f. [Ca2+]
rest
i
=∼50 to 100 nm), veratridine produced only a small cortical [Ca2+]
i
transient. This increased in size and spatial distribution at [Ca2+]
o
= 50 μm of 1 mm. Interestingly with unusually high yet nontoxic [Ca2+]
o
= 10 mm, [Ca2+]
i
transients were much delayed and also reduced, as is trichocyst exocytosis. We interpret our results as follows. (i) With
[Ca2+]
o
= 30 nm, the restricted residual response observed is due to Ca2+ mobilization from subplasmalemmal stores. (ii) With moderate [Ca2+]
o
= 50 μm to 1 mm, the established membrane labilizing effect of veratridine may activate not only subplasmalemmal stores but also Ca2+
o influx from the medium via so far unidentified (anteriorly enriched) channels. Visibility of these phenomena is best in tl
cells, where free docking sites allow for rapid Ca2+ spread, and least in 7S cells, whose perfectly assembled docking sites may ``consume' a large part of the [Ca2+]
i
increase. (iii) With unusually high [Ca2+]
o
, mobilization of cortical stores and/or Ca2+
o
influx may be impeded by the known membrane stabilizing effect of Ca2+
o
counteracting the labilizing/channel activating effect of veratridine. (iv) We show these effects to be reversible, and,
hence, not to be toxic side-effects, as confirmed by retention of injected calcein.
(v) Finally, Mn2+ entry during veratridine stimulation, documented by Fura-2 fluorescence quenching, may indicate activation of unspecific
Me2+ channels by veratridine. Our data have some bearing on analysis of other cells, notably neurons, whose response to veratridine
is of particular and continous interest.
Received: 8 December 1998/Revised: 2 March 1999 相似文献
7.
Caffeine causes a [Ca2+]
i
increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca2+]rest
i
∼50 to 80 nm, [Ca2+]act
i
rises within ≤3 sec to 500 (trichocyst-free strain tl) or 220 nm (nondischarge strain nd9–28°C) in the cortex. Rapid confocal analysis of wildtype cells (7S) showed only a 2-fold cortical
increase within 2 sec, accompanied by trichocyst exocytosis and a central Ca2+ spread during the subsequent ≥2 sec. Chelation of Ca2+
o
considerably attenuated [Ca2+]
i
increase. Therefore, caffeine may primarily mobilize cortical Ca2+ pools, superimposed by Ca2+ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst
contents to decondense internally (Ca2+-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small
extent, but with increasing frequency as [Ca2+]
i
signals were reduced by [Ca2+]
o
chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components
(without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca2+]
i
signal was generated by caffeine stimulation (with Ca2+
i
and Ca2+
o
available). We conclude the following. (i) Caffeine can mobilize Ca2+ from cortical stores independent of the presence of Ca2+
o
. (ii) To yield adequate signals for normal exocytosis, Ca2+ release and Ca2+ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca2+]
i
increase entails caffeine-mediated access of Ca2+ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation
in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply
different threshold [Ca2+]
i
-values for membrane fusion and contents discharge.
Received: 23 May 1997/Revised: 18 August 1997 相似文献
8.
Using 5% ethanol as a deciliating agent, 20 mm colchicine to prevent reciliation and 1 mm amiloride to affect ion fluxes in Paramecium we examined the compartmentation and function of Ca2+ fluxes employing the biosynthesis of cGMP and the stereotypic swimming behavior as indicators for Ca2+ entry. As a function of extracellular Ca2+
Paramecia responded to colchicine and amiloride with a short-lived ciliary augmentation (fast swimming) which indicated hyperpolarization,
and formation of cGMP, i.e., the reported hyperpolarization-activated Ca2+ inward current in the somatic membrane is coupled to intracellular generation of cGMP. This is comparable to the coupling
of the depolarization-activated, ciliary Ca2+ inward current and ciliary cGMP formation.
Ethanol-deciliated cells and ethanol-treated, yet ciliated control cells did not respond to a depolarization with backward
swimming or formation of cGMP. Both responses recovered with similar kinetics. A persistent effect of an ethanol exposure
on the axonemal apparatus or on guanylyl cyclase activity of ciliated control cells was excluded using permeabilized cells
and cell-free enzyme, respectively. Further, in the presence of 20 mm colchicine ethanol-treated cells only recovered the depolarization-dependent avoiding reaction whereas the formation of cGMP
remained depressed, i.e., the drug dissected both responses. Similarly, ethanol exposure of Paramecia did not affect the fast swimming response towards the hyperpolarizing agent amiloride whereas the cGMP formation was abrogated
and recovered over a period of 7 hr, i.e., amiloride dissected the hyperpolarization-elicited behavioral response from the
intracellular cGMP formation.
The data demonstrate that in Paramecium depolarization- and hyperpolarization-stimulated behavioral responses and cGMP formation are not coupled. The behavioral
changes are triggered by smaller Ca2+ inward currents than the formation of intracellular cGMP.
Received: 8 August 1996/Revised: 15 November 1996 相似文献
9.
We investigated the cytosolic free calcium concentration ([Ca2+]i) of leech Retzius neurons in situ while varying the extracellular Ca2+ concentration via the bathing solution ([Ca2+]B). Changing [Ca2+]B had only an effect on [Ca2+]i if the cells were depolarized by raising the extracellular K+ concentration. Surprisingly, raising [Ca2+]B from 2 to 10 mm caused a decrease in [Ca2+]i, and an increase was evoked by reducing [Ca2+]B to 0.1 mm. These changes were not due to shifts in membrane potential. At low [Ca2+]B moderate membrane depolarizations were sufficient to evoke a [Ca2+]i increase, while progressively larger depolarizations were necessary at higher [Ca2+]B. The changes in the relationship between [Ca2+]i and membrane potential upon varying [Ca2+]B could be reversed by changing extracellular pH. We conclude that [Ca2+]B affects [Ca2+]i by modulating Ca2+ influx through voltage-dependent Ca2+ channels via the electrochemical Ca2+ gradient and the surface potential at the extracellular side of the plasma membrane. These two parameters are affected in
a counteracting way: Raising the extracellular Ca2+ concentration enhances the electrochemical Ca2+ gradient and hence Ca2+ influx, but it attenuates Ca2+ channel activity by shifting the extracellular surface potential to the positive direction, and vice versa.
Received: 23 January 2001/Revised: 23 June 2001 相似文献
10.
This study examined [Ca2+]i oscillations in the human salivary gland cell lines, HSY and HSG. Relatively low concentrations of carbachol (CCh) induced
oscillatory, and higher [CCh] induced sustained, steady-state increases in [Ca2+]i and K
Ca currents in both cell types. Low IP3, but not thapsigargin (Tg), induced [Ca2+]i oscillations, whereas Tg blocked CCh-stimulated [Ca2+]i oscillations in both cell types. Unlike in HSG cells, removal of extracellular Ca2+ from HSY cells (i) did not affect CCh-stimulated [Ca2+]i oscillations or internal Ca2+ store refill, and (ii) converted high [CCh]-induced steady-state increase in [Ca2+]i into oscillations. CCh- or thapsigargin-induced Ca2+ influx was higher in HSY, than in HSG, cells. Importantly, HSY cells displayed relatively higher levels of sarcoendoplasmic
reticulum Ca2+ pump (SERCA) and inositoltrisphosphate receptors (IP3Rs) than HSG cells.
These data demonstrate that [Ca2+]i oscillations in both HSY and HSG cells are primarily determined by the uptake of Ca2+ from, and release of Ca2+ into, the cytosol by the SERCA and IP3R activities, respectively. In HSY cells, Ca2+ influx does not acutely contribute to this process, although it determines the steady-state increase in [Ca2+]i. In HSG cells, [Ca2+]i oscillations directly depend on Ca2+ influx; Ca2+ coming into the cell is rapidly taken up into the store and then released into the cytosol. We suggest that the differences
in the mechanism of [Ca2+]i oscillations HSY and HSG cells is related to their respective abilities to recycle internal Ca2+ stores.
Received: 30 October 2000/Revised: 26 February 2001 相似文献
11.
Lung lamellar bodies maintain an acidic interior by an energy-dependent process. The acidic pH may affect the packaging of
surfactant phospholipids, processing of surfactant proteins, or surfactant protein A-dependent lipid aggregation. The electron-probe
microanalysis of lamellar body elemental composition has previously suggested that lamellar bodies contain high levels of
calcium some of which may be in ionic form. In this study, we investigated the Ca2+ uptake characteristics in isolated lung lamellar bodies. The uptake of Ca2+ was measured by monitoring changes in the fluorescence of Fluo-3, a Ca2+ indicator dye. The uptake of Ca2+ in lamellar bodies was ATP-dependent and increased with increasing concentrations of Ca2+. At 100 nm Ca2+, the uptake was almost completely inhibited by bafilomycin A1, a selective inhibitor of vacuolar type H+-ATPase, or by NH4Cl, which raises the lamellar body pH, suggesting that the pH gradient regulates the uptake. The uptake of Ca2+ increased as the Ca2+ concentration was increased, but the relative contribution of bafilomycin A1-sensitive uptake decreased. At 700 nm, it comprised only 20% of the total uptake. These results suggest the presence of additional mechanism(s) for uptake at higher
Ca2+ concentrations. At 700 nm Ca2+, the rate and extent of uptake were lower in the absence of K+ than in the presence of K+. The inhibitors of Ca2+-activated K+-channels, tetraethylammonium, Penitrem A, and 4-aminopyridine, also inhibited the K+-dependent Ca2+ uptake at 700 nm Ca2+. Thus the uptake of Ca2+ in isolated lung lamellar bodies appears to be regulated by two mechanisms, (i) the H+-gradient and (ii) the K+ transport across the lamellar body membrane. We speculate that lamellar bodies accumulate Ca2+ and contribute to regulation of cytosolic Ca2+ in type II cells under resting and stimulated conditions.
Received: 18 August 1999/Revised: 9 November 1999 相似文献
12.
The plasma membrane calcium pump, which ejects Ca2+ from the cell, is regulated by calmodulin. In the absence of calmodulin, the pump is relatively inactive; binding of calmodulin
to a specific domain stimulates its activity. Phosphorylation of the pump with protein kinase C or A may modify this regulation.
Most of the regulatory functions of the enzyme are concentrated in a region at the carboxyl terminus. This region varies substantially
between different isoforms of the pump, causing substantial differences in regulatory properties. The pump shares some motifs
of the carboxyl terminus with otherwise unrelated proteins: The calmodulin-binding domain is a modified IQ motif (a motif
which is present in myosins) and the last 3 residues of isoform 4b are a PDZ target domain. The pump is ubiquitous, with isoforms
1 and 4 of the pump being more widely distributed than 2 and 3. In some kinds of cells isoform 1 or 4 is missing, and is replaced
by another isoform.
Received: 26 January 1998/Revised: 6 April 1998 相似文献
13.
Péter M Varga Z Hajdu P Gáspár R Damjanovich S Horjales E Possani LD Panyi G 《The Journal of membrane biology》2001,179(1):13-25
We investigated the cytosolic free Ca2+ concentration ([Ca2+]i) of leech Retzius neurons in situ while varying the extracellular and intracellular pH as well as the extracellular ionic
strength. Changing these parameters had no significant effect on [Ca2+]i when the membrane potential of the cells was close to its resting value. However, when the cells were depolarized by raising
the extracellular K+ concentration or by applying the glutamatergic agonist kainate, extracellular pH and ionic strength markedly affected [Ca2+]i, whereas intracellular pH changes appeared to have virtually no effect. An extracellular acidification decreased [Ca2+]i, while alkalinization or reduction of the ionic strength increased it. Correspondingly, [Ca2+]i also increased when the kainate-induced extracellular acidification was reduced by raising the pH-buffering capacity. At
low extracellular pH, the membrane potential to which the cells must be depolarized to evoke a detectable [Ca2+]i increase was shifted to more positive values, and it moved to more negative values at high pH. We conclude that in leech
Retzius neurons extracellular pH, but not intracellular pH, affects [Ca2+]i by modulating Ca2+ influx through voltage-dependent Ca2+ channels. The results suggest that this modulation is mediated primarily by shifts in the surface potential at the extracellular
side of the plasma membrane.
Received: 23 January 2001/Revised: 15 June 2001 相似文献
14.
To assess the activation of the charybdotoxin-insensitive K+ channel responsible for Regulatory Volume Decrease (RVD) after substantial volume increases, we measured intracellular pH
(pH
i
), intracellular calcium ([Ca2+]
i
) and inhibitors of kinases and phosphoprotein phosphatases in guinea pig jejunal villus enterocytes in response to volume
changes. Fluorescence spectroscopy was used to measure pH
i
and [Ca2+]
i
of cells in suspension, loaded with 2,7,bis-carboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively, and cell volume
was assessed using electronic cell sizing. A modest 7% volume increase or substantial 15 to 20% volume increase caused [Ca2+]
i
to increase proportionately but the 7% increase caused alkalinization while the larger increases resulted in acidification
of ≃0.14 pH units. Following a 15% volume increase, 1-N-0-bis (5-isoquinoline-sulfonyl)-N-methyl-l-4-phenyl-piperazine (KN-62, 50 μm), an inhibitor of Ca2+/calmodulin kinase II, blocked RVD. Gramicidin (0.5 μm) bypassed this inhibition suggesting that the K+ channel had been affected by the KN-62. RVD after a modest 7% volume increase was not influenced by KN-62 unless the cell
was acidified. Okadaic acid, an inhibitor of phosphoprotein phosphatases 1 and 2A, accelerated RVD after a 20% volume increase;
inhibition of RVD generated by increasing the K+ gradient was bypassed by okadaic acid. Tyrosine kinase inhibitor, genistein (100 μm) had no effect on RVD after 20% volume increases. We conclude that activation of charybdotoxin-insensitive K+ channels utilized for RVD after substantial (>7%) `nonphysiological' volume increases requires phosphorylation mediated by
Ca2+/calmodulin kinase II and that increases in cytosolic acidification rather than larger increases in [Ca2+]
i
are a critical determinant of this activation.
Received: 30 March 1999/Revised: 6 July 1999 相似文献
15.
We combined widely different biochemical methods to analyze proteins of the cell surface of P. tetraurelia since so far one can isolate only a subfraction of cell membrane vesicles enriched in the GPI-anchored surface antigens (``immoblization'
or ``i-AGs'). We also found that i-AGs may undergo partial degradation by endogenous proteases. Genuine intrinsic membrane
proteins were recognized particularly with lipophilic 5-[125I]-iodonaphthalene-1-azide (INA) labeling which reportedly ``sees' integral proteins and cytoplasmic cell membrane-associated
proteins. With INA (+DTT), bands of ≤55 kDa were similar as with hydrophilic iodogen (+DTT), but instead of large size bands
including i-AGs, a group of 122, 104 and 94 kDa appeared. Several bands of the non i-AG type are compatible with integral
(possibly oligomeric) or associated proteins of the cell membrane of established molecular identity, as we discuss. In summary,
we can discriminate between i-AGs and some functionally important minor cell membrane components. Our methodical approach
might be relevant also for an analysis of some related protozoan parasites.
Received: 5 April 1999/Revised: 19 July 1999 相似文献
16.
X. Wu H. Yang P. Iserovich J. Fischbarg P.S. Reinach 《The Journal of membrane biology》1997,158(2):127-136
The relationship between relative cell volume and time-dependent changes in intracellular Ca2+ concentration ([Ca2+]
i
) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light
scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic
time (τ
vr
) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca2+]
i
initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell
volume recovery and [Ca2+]
i
transients were inhibited by either: (a) Ca2+-free medium; (b) 5 mm Ni2+ (inhibitor of plasmalemma Ca2+ influx); (c) 10 μm cyclopiazonic acid, CPA (which causes depletion of intracellular Ca2+ content); or (d) 100 μm ryanodine (inhibitor of Ca2+ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca2+ influx and the emptying of intracellular Ca2+ stores during the [Ca2+]
i
transients, Mn2+ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn2+ permeability 6-fold. However, Mn2+ permeability remained unchanged during exposure to either: 1.100 μm ryanodine; 2.10 μm CPA and 100 μm ryanodine. This report for the first time documents the time dependence of the components of the [Ca2+]
i
transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca2+ release from an intracellular store leads to a subsequent increase in plasmalemma Ca2+ influx, and that both are required for cells to undergo RVD.
Received: 7 November 1996/Revised: 6 January 1997 相似文献
17.
18.
In cystic fibrosis, the mutation of the CFTR protein causes reduced transepithelial Cl− secretion. As recently proposed, beside its role of Cl− channel, CFTR may regulate the activity of other channels such as a Ca2+-activated Cl− channel. Using a calcium imaging system, we show, in adenovirus-CFTR infected Chinese Hamster Ovary (CHO) cell monolayers,
that CFTR can act as a regulator of intracellular [Ca2+]
i
([Ca2+]
i
), involving purino-receptors. Apical exposure to ATP or UTP produced an increase in ([Ca2+]
i
in noninfected CHO cell monolayers (CHO-WT), in CHO monolayers infected with an adenovirus-CFTR (CHO-CFTR) or infected with
an adenovirus-LacZ (CHO-LacZ). The transient [Ca2+]
i
increase produced by ATP or UTP could be mimicked by activation of CFTR with forskolin (20 μm) in CHO-CFTR confluent monolayers. However, forskolin had no significant effect on [Ca2+]
i
in noninfected CHO-WT or in CHO-LacZ cells. Pretreatment with purino-receptor antagonists such as suramin (100 μm) or reactive blue-2. (100 μm), and with hexokinase (0.28 U/mg) inhibited the [Ca2+]
i
response to forskolin in CHO-CFTR infected cells. Taken together, our experiments provide evidence for purino-receptor activation
by ATP released from the cell and regulation of [Ca2+]
i
by CFTR in CHO epithelial cell membranes.
Received: 5 April 1999/Revised: 28 June 1999 相似文献
19.
The purine nucleotide GTP causes a complex behavioral response and two distinct electrophysiological responses in the ciliated
protozoan Paramecium tetraurelia. One of the two electrophysiological responses is an oscillating current that is responsible for the repeated backward swimming
episodes that constitute the behavioral response to GTP. The second electrophysiological response is a sustained current whose
relationship to the first is unknown. Here we show that the purine nucleotide XTP can completely block both the behavioral
response to GTP and its associated oscillating current, but not the sustained current induced by GTP. Notably, XTP alone causes
a sustained current similar to that induced by GTP. We believe the data support the notion that P. tetraurelia possesses two distinct signal transduction pathways sensitive to purine nucleotides: one specific for GTP that leads to oscillating
currents and behavior, and a second pathway activated by GTP and other purine nucleotides that leads to a sustained current.
Received: 22 August 1997/Revised: 20 January 1998 相似文献
20.
Plant growth requires a continuous supply of intracellular solutes in order to drive cell elongation. Ion fluxes through
the plasma membrane provide a substantial portion of the required solutes. Here, patch clamp techniques have been used to
investigate the electrical properties of the plasma membrane in protoplasts from the rapid growing tip of maize coleoptiles.
Inward currents have been measured in the whole cell configuration from protoplasts of the outer epidermis and from the cortex.
These currents are essentially mediated by K+ channels with a unitary conductance of about 12 pS. The activity of these channels was stimulated by negative membrane voltage
and inhibited by extracellular Ca2+ and/or tetraethylammonium-CI (TEA). The kinetics of voltage- and Ca2+-gating of these channels have been determined experimentally in some detail (steady-state and relaxation kinetics). Various
models have been tested for their ability to describe these experimental data in straightforward terms of mass action. As
a first approach, the most appropriate model turned out to consist of an active state which can equilibrate with two inactive
states via independent first order reactions: a fast inactivation/activation by Ca2+-binding and -release, respectively (rate constants >>103 sec−1) and a slower inactivation/activation by positive/negative voltage, respectively (voltage-dependent rate constants in the
range of 103 sec−1).
With 10 mm K+ and 1 mm Ca2+ in the external solution, intact coleoptile cells have a membrane voltage (V) of −105 ± 7 mV. At this V, the density and open probability of the inward-rectifying channels is sufficient to mediate K+ uptake required for cell elongation. Extracellular TEA or Ca2+, which inhibit the K+ inward conductance, also inhibit elongation of auxin-depleted coleoptile segments in acidic solution. The comparable effects
of Ca2+ and TEA on both processes and the similar Ca2+ concentration required for half maximal inhibition of growth (4.3 mm Ca2+) and for conductance (1.2 mm Ca2+) suggest that K+ uptake through the inward rectifier provides essential amounts of solute for osmotic driven elongation of maize coleoptiles.
Received: 6 June 1995/Revised: 12 September 1995 相似文献