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1.
In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl− secretion to secretagogues acting via cAMP. Using a Ca2+ imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca2+]
i
via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase
in [Ca2+]
i
in normal (16HBE14o− cell line and primary lung culture) and in cystic fibrosis (CFTE29o− cell line) human airway epithelia. The potency order of nucleotides on [Ca2+]
i
variation was UTP ≫ ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca2+]
i
response could be mimicked by activation of CFTR with forskolin (20 μm) in a temperature-dependent manner. In 16HBE14o− cells, the forskolin-induced [Ca2+]
i
response increased with increasing temperature. In CFTE29o− cells, forskolin had no effect on [Ca2+]
i
at body temperature-forskolin-induced [Ca2+]
i
response in CF cells could only be observed at low experimental temperature (14°C) or when cells were cultured at 26°C instead
of 37°C. Pretreatment with CFTR channel blockers glibenclamide (100 μm) and DPC (100 μm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 μm), inhibited the forskolin [Ca2+]
i
response. Together, these results demonstrate that once activated, CFTR regulates [Ca2+]
i
by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia.
Received: 3 April 2000/Revised: 30 June 2000 相似文献
2.
We evaluated mechanisms which mediate alterations in intracellular biochemical events in response to transient mechanical
stimulation of colonic smooth muscle cells. Cultured myocytes from the circular muscle layer of the rabbit distal colon responded
to brief focal mechanical deformation of the plasma membrane with a transient increase in intracellular calcium concentration
([Ca2+]
i
) with peak of 422.7 ± 43.8 nm above an average resting [Ca2+]
i
of 104.8 ± 10.9 nm (n= 57) followed by both rapid and prolonged recovery phases. The peak [Ca2+]
i
increase was reduced by 50% in the absence of extracellular Ca2+, while the prolonged [Ca2+]
i
recovery was either abolished or reduced to ≤15% of control values. In contrast, no significant effect of gadolinium chloride
(100 μm) or lanthanum chloride (25 μm) on either peak transient or prolonged [Ca2+]
i
recovery was observed. Pretreatment of cells with thapsigargin (1 μm) resulted in a 25% reduction of the mechanically induced peak [Ca2+]
i
response, while the phospholipase C inhibitor U-73122 had no effect on the [Ca2+]
i
transient peak. [Ca2+]
i
transients were abolished when cells previously treated with thapsigargin were mechanically stimulated in Ca2+-free solution, or when Ca2+ stores were depleted by thapsigargin in Ca2+-free solution. Pretreatment with the microfilament disrupting drug cytochalasin D (10 μm) or microinjection of myocytes with an intracellular saline resulted in complete inhibition of the transient. The effect
of cytochalasin D was reversible and did not prevent the [Ca2+]
i
increases in response to thapsigargin. These results suggest a communication, which may be mediated by direct mechanical
link via actin filaments, between the plasma membrane and an internal Ca2+ store.
Received: 24 March 1997/Revised: 21 July 1997 相似文献
3.
Using spectrofluorescence imaging of fura-2 loaded renal A6 cells, we have investigated the generation of the cytosolic Ca2+ signal in response to osmotic shock and localized membrane stretch. Upon hypotonic exposure, the cells began to swell prior
to a transient increase in [Ca2+]
i
and the cells remained swollen after [Ca2+]
i
had returned towards basal levels. Exposure to 2/3rd strength Ringer produced a cell volume increase within 3 min, followed
by a slow regulatory volume decrease (RVD). The hypotonic challenge also produced a transient increase in [Ca2+] after a delay of 22 sec. Both the RVD and [Ca2+]
i
response to hypotonicity were inhibited in a Ca2+-free bathing solution and by gadolinium (10 μm), an inhibitor of stretch-activated channels. Stretching the membrane by application of subatmospheric pressure (-2 kPa)
inside a cell-attached patch-pipette induced a similar global increase in [Ca2+]
i
as occurred after hypotonic shock. A stretch-sensitive [Ca2+]
i
increase was also observed in a Ca2+-free bathing solution, provided the patch-pipette contained Ca2+. The mechanosensitive [Ca2+]
i
response was by gadolinium (10 μm) or Ca2+-free pipette solutions, even when Ca2+ (2 mm) was present in the bath. Long-term (>10 min) pretreatment of the cells with thapsigargin inhibited the [Ca2+]
i
response to hypotonicity. These results provide evidence that cell swelling or mechanical stimulation can activate a powerful
amplification system linked to intracellular Ca2+ release mechanisms.
Received: 3 August 1998/Revised: 19 November 1998 相似文献
4.
We studied the relationship between changes in intracellular pH (pH
i
), intracellular Ca2+([Ca2+]
i
) and charybdotoxin sensitive (CTX) maxi-K+ channels occurring after modest `physiological' swelling in guinea pig jejunal villus enterocytes. Villus cell volume was
assessed by electronic cell sizing, and pH
i
and [Ca2+]
i
by fluorescence spectroscopy with 2,7, biscarboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively. In a slightly (0.93
× isotonic) hypotonic medium, villus cells swelled to the same size they would reach during d-glucose or l-alanine absorption; the subsequent Regulatory Volume Decrease (RVD) was prevented by CTX. After the large volume increase
in a more hypotonic (0.80 × isotonic) medium, RVD was unaffected by CTX. After modest swelling associated with 0.93 × isotonic
dilution, the pH
i
alkalinized but N-5-methyl-isobutyl amiloride (MIA) prevented this ΔpH
i
and the subsequent RVD. Even in the presence of MIA, alkalinization with added NH4Cl permitted complete RVD which could be inhibited by CTX. The rate of 86Rb efflux which also increased after this 0.93 × isotonic dilution was inhibited an equivalent amount by CTX, MIA or Na+-free medium. Modest swelling transiently increased [Ca2+]
i
and Ca2+-free medium or blocking alkalinization by MIA or Na+-free medium diminished this transient increase an equivalent amount. RVD after modest swelling was prevented in Ca2+-free medium but alkalinization still occurred. After large volume increases, alkalinization of cells increased [Ca2+]
i
and volume changes became sensitive to CTX. We conclude that both alkalinization of pH
i
and increased [Ca2+]
i
observed with `physiological' volume increase are essential for the activation of CTX-sensitive maxi-K+ channels required for RVD.
Received: 30 March 1999/Revised: 6 July 1999 相似文献
5.
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
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.
Stimulation with leukotriene D4 (LTD4) (3–100 nm) induces a transient increase in the free intracellular Ca2+ concentration ([Ca2+]
i
) in Ehrlich ascites tumor cells. The LTD4-induced increase in [Ca2+]
i
is, however, significantly reduced in Ca2+-free medium (2 mm EGTA), and under these conditions stimulation with a low LTD4 concentration (3 nm) does not result in any detectable increase in [Ca2+]
i
. Addition of LTD4 (3–100 nm) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium.
The LTD4-induced (100 nm) acceleration of the RVD response is also seen in Ca2+-free medium and also at 3 nm LTD4, indicating that LTD4 can open K+- and Cl−-channels without any detectable increase in [Ca2+]
i
. Buffering cellular Ca2+ with BAPTA almost completely blocks the LTD4-induced (100 nm) acceleration of the RVD response. Thus, the reduced [Ca2+]
i
level after BAPTA-loading or buffering of [Ca2+]
i
seems to inhibit the LTD4-induced stimulation of the RVD response even though the LTD4-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca2+]
i
. The LTD4 receptor antagonist L649,923 (1 μm) completely blocks the LTD4-induced increase in [Ca2+]
i
and inhibits the RVD response as well as the LTD4-induced acceleration of the RVD response. When the LTD4 receptor is desensitized by preincubation with 100 nm LTD4, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD4 plays a role in the activation of the RVD response. LTD4 seems to activate K+ and Cl− channels via stimulation of a LTD4 receptor with no need for a detectable increase in [Ca2+]
i
.
Received: 25 September 1995/Revised: 25 January 1996 相似文献
8.
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
9.
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
10.
S. Pedersen E.K. Hoffmann C. Hougaard N.K. Jørgensen G.B. Wybrandt I.H. Lambert 《The Journal of membrane biology》1997,155(1):61-73
Stimulation of Ehrlich ascites tumor cells with leukotriene D4 (LTD4) within the concentration range 1–100 nm leads to a concentration-dependent, transient increase in the intracellular, free Ca2+ concentration, [Ca2+]
i
. The Ca2+ peak time, i.e., the time between addition of LTD4 and the highest measured [Ca2+]
i
value, is in the range 0.20 to 0.21 min in ten out of fourteen independent experiments. After addition of a saturating concentration
of LTD4 (100 nm), the highest measured increase in [Ca2+]
i
in Ehrlich cells suspended in Ca2+-containing medium is 260 ± 14 nm and the EC50 value for LTD4-induced Ca2+ mobilization is estimated at 10 nm. Neither the peptido-leukotrienes LTC4 and LTE4 nor LTB4 are able to mimic or block the LTD4-induced Ca2+ mobilization, hence the receptor is specific for LTD4. Removal of Ca2+ from the experimental buffer significantly reduces the size of the LTD4-induced increase in [Ca2+]
i
. Furthermore, depletion of the intracellular Ins(1,4,5)P3-sensitive Ca2+ stores by addition of the ER-Ca2+-ATPase inhibitor thapsigargin also reduces the size of the LTD4-induced increase in [Ca2+]
i
in Ehrlich cells suspended in Ca2+-containing medium, and completely abolishes the LTD4-induced increase in [Ca2+]
i
in Ehrlich cells suspended in Ca2+-free medium containing EGTA. Thus, the LTD4-induced increase in [Ca2+]
i
in Ehrlich cells involves an influx of Ca2+ from the extracellular compartment as well as a release of Ca2+ from intracellular Ins(1,4,5)P3-sensitive stores. The Ca2+ peak times for the LTD4-induced Ca2+ influx and for the LTD4-induced Ca2+ release are recorded in the time range 0.20 to 0.21 min in four out of five experiments and in the time range 0.34 to 0.35
min in six out of eight experiments, respectively. Stimulation with LTD4 also induces a transient increase in Ins(1,4,5)P3 generation in the Ehrlich cells, and the Ins(1,4,5)P3 peak time is recorded in the time range 0.27 to 0.30 min. Thus, the Ins(1,4,5)P3 content seems to increase before the LTD4-induced Ca2+ release from the intracellular stores but after the LTD4-induced Ca2+ influx. Inhibition of phospholipase C by preincubation with U73122 abolishes the LTD4-induced increase in Ins(1,4,5)P3 as well as the LTD4-induced increase in [Ca2+]
i
, indicating that a U73122-sensitive phospholipase C is involved in the LTD4-induced Ca2+ mobilization in Ehrlich cells. The LTD4-induced Ca2+ influx is insensitive to verapamil, gadolinium and SK&F 96365, suggesting that the LTD4-activated Ca2+ channel in Ehrlich cells is neither voltage gated nor stretch activated and most probably not receptor operated. In conclusion,
LTD4 acts in the Ehrlich cells via a specific receptor for LTD4, which upon stimulation initiates an influx of Ca2+, through yet unidentified Ca2+ channels, and an activation of a U73122-sensitive phospholipase C, Ins(1,4,5)P3 formation and finally release of Ca2+ from the intracellular Ins(1,4,5)P3-sensitive stores.
Received: 9 February 1996/Revised: 15 August 1996 相似文献
11.
O. Strauß K. Steinhausen S. Mergler F. Stumpff M. Wiederholt 《The Journal of membrane biology》1999,169(3):141-153
This combined study of patch-clamp and intracellular Ca2+ ([Ca2+]
i
) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca2+-dependent Cl− channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca2+]
i
and activation of Cl− currents. In contrast, intracellular application of Ca2+ (10 μm) only induced transient activation of Cl− currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca2+ and to the blocker of I
CRAC, La3+ (10 μm), despite the fact that both maneuvers led to a decline in [Ca2+]
i
. The InsP3-induced rise in Cl− conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca2+ stores or by removal of Ca2+ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca2+-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca2+/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl−-conductance in 50% of the cells investigated without affecting [Ca2+]
i
. Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca2+]
i
and Cl− conductance. In summary, elevation of [Ca]
i
by InsP3 leads to activation of Cl− channels involving cytosolic Ca2+ stores and Ca2+ influx from extracellular space. Tyrosine kinases are essential for the Ca2+-independent maintenance of this conductance.
Received: 15 October 1998/Revised: 3 March 1999 相似文献
12.
The presence and localization of voltage-gated Ca2+ channels of L-type were investigated in intestinal cells of the Atlantic cod. Enterocytes were loaded with the fluorescent
Ca2+ probe, fure-2/AM and changes in intracellular Ca2+ concentrations ([Ca2+]
i
) were measured, in cell suspensions, in the presence of high potassium levels (100 mm), BAY K-8644 (5 μm), nifedipine (5 μm) or ω-conotoxin (1 μm). L-type Ca2+ channels were visualized on intestinal sections using the fluorescent dihydropyridine (-)-STBodipy.
Depolarization of the plasma membrane produced a rapid (within 5 sec) and transient (at basal levels after 21 sec) increase
in [Ca2+]
i
. BAY K-8644 increased the [Ca2+]
i
by 7.2%. Cells in a Ca2+-free buffer increased [Ca2+]
i
after addition of 10 mm Ca2+, and this increase was abolished by nifedipine in both depolarizing and normal medium but not by ω-conotoxin. Single cell
experiments using video microscopy revealed that enterocytes remained polarized several hours after preparation and that the
Ca2+ entry and extrusion occurred at specific and different regions of the enterocyte outer membrane. Fluorescent staining of
L-type Ca2+ channels in the intestinal mucosa showed the most intense staining at the brushborder membrane.
These results demonstrate the presence of voltage gated L-type Ca2+ channels in enterocytes from the Atlantic cod. The channels are mainly located at the apical side of the cells, and there
is a polarized uptake of Ca2+ into the enterocytes. This suggests that the L-type Ca2+ channels are involved in the transcellular Ca2+ entry into the enterocytes.
Received: 21 August 1997/Revised: 15 April 1998 相似文献
13.
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
14.
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
15.
C. Hirono M. Sugita K. Furuya S. Yamagishi Y. Shiba 《The Journal of membrane biology》1998,164(2):197-203
Isoproterenol (IPR) and 8-(4-chlorophenylthio)-cyclic AMP (cpt-cAMP) enhanced carbachol (CCh)-induced fluid secretion from
rat parotid glands, but had no effect by themselves. The enhancement by IPR was blocked by propranolol. In dispersed parotid
acinar cells, IPR and cpt-cAMP potentiated CCh-induced K+ and Cl− currents (I
K and I
Cl). IPR at the concentration of 0.1 μm significantly potentiated the CCh-induced increase in intracellular Ca2+ concentration ([Ca2+]
i
), but 1 mm cpt-cAMP did not. The incidence of the potentiation by IPR in CCh-induced Mn2+ entry was 31% and that by cpt-cAMP was 21%. The potentiation by IPR in the ionic currents and the [Ca2+]
i
was suppressed by propranolol. These results suggest that the CCh-induced fluid secretion from rat parotid glands is enhanced
by IPR through the potentiation of I
K and I
Cl mainly by the increased cyclic AMP level and partially by the potentiated Ca2+ influx and [Ca2+]
i
increase, and that IPR is more effective than cpt-cAMP in the enhancement of the CCh-induced [Ca2+]
i
increase.
Received: 6 October 1997/Revised: 16 April 1998 相似文献
16.
Differential desensitization of Ca2+ mobilization and vasoconstriction by ET(A) receptors in the gerbil spiral modiolar artery 总被引:2,自引:0,他引:2
Endothelins are known to be among the most potent endogenous vasoconstrictors. Vasoconstriction of the spiral modiolar artery,
which supplies the cochlea, may be implicated in hearing loss and tinnitus. The purpose of the present study was to determine
whether the spiral modiolar artery responds to endothelin, whether a change in the cytosolic Ca2+ concentration ([Ca2+]i) mediates the response and which endothelin receptors are present. The vascular diameter and [Ca2+]i were measured simultaneously by videomicroscopy and microfluorometry in the isolated spiral modiolar artery from the gerbil.
ET-1 induced a transient [Ca2+]i increase and a strong and long-lasting vasoconstriction. The transient [Ca2+]i increase underwent rapid desensitization, was independent of extracellular Ca2+ and inhibited by the IP3-receptor blocker (75 μm) 2-aminoethoxydiphenyl borate (2-APB) and by depletion of Ca2+ stores with 10−6
m thapsigargin. In contrast, the vasoconstriction displayed no comparable desensitization. The initial vasoconstriction was
independent of extracellular Ca2+ but maintenance of the constriction depended on the presence of extracellular Ca2+. The half-maximal concentration values (EC
50) for the agonists ET-1, ET-3 and sarafotoxin S6c were 0.8 nm, >10 nm and >100 nm, respectively. Affinity constants for the antagonists BQ-123 and BQ-788 were 24 nm and 77 nm, respectively. These observations demonstrate that ET-1 mediates a vasoconstriction of the gerbil spiral modiolar artery
via ETA receptors and an IP3 receptor-mediated release of Ca2+ from thapsigargin-sensitive Ca2+ stores. The marked difference in desensitization between Ca2+ mobilization and vasoconstriction suggests that Ca2+ mobilization is not solely responsible for the vasoconstriction and that other signaling mechanisms must be present.
Received: 4 January 2001/Revised: 23 April 2001 相似文献
17.
S.F. Pedersen J. Prenen G. Droogmans E.K. Hoffmann B. Nilius 《The Journal of membrane biology》1998,163(2):97-110
A Ca2+-activated (I
Cl,Ca) and a swelling-activated anion current (I
Cl,vol) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying
currents were activated by an increase in the free intracellular calcium concentration ([Ca2+]
i
), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular
Cl− concentration. I
Cl,Ca current density increased with increasing [Ca2+]
i
, and this current was abolished by lowering [Ca2+]
i
to <1 nm using 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA). In contrast, activation of I
Cl,vol did not require an increase in [Ca2+]
i
. The kinetics of I
Cl,Ca and I
Cl,vol were different: at depolarized potentials, I
Cl,Ca as activated in a [Ca2+]
i
- and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, I
Cl,vol exhibited time- and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials.
The deactivation of I
Cl,vol was dependent on the extracellular Mg2+ concentration. The anion permeability sequence for both currents was I
− > Cl− > gluconate. I
Cl,Ca was inhibited by niflumic acid (100 μm), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 μm) and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS, 100 μm), niflumic acid being the most potent inhibitor. In contrast, I
Cl,vol was unaffected by niflumic acid (100 μm), but abolished by tamoxifen (10 μm). Thus, in Ehrlich cells, separate chloride currents, I
Cl,Ca and I
Cl,vol, are activated by an increase in [Ca2+]
i
and by cell swelling, respectively.
Received: 12 November 1997/Revised: 5 February 1998 相似文献
18.
The calcium indicator fura-2 was used to study the effect of hypotonic solutions on the intracellular calcium concentration,
[Ca2+]
i
, in a human osteoblast-like cell line. Decreasing the tonicity of the extracellular solution to 50% leads to an increase
in [Ca2+]
i
from ∼150 nm up to 1.3 μm. This increase in [Ca2+]
i
was mainly due to an influx of extracellular Ca2+ since removing of extracellular Ca2+ reduced this increase to ∼250 nm. After cell swelling most of the cells were able to regulate their volume to the initial level within 800 sec. The whole-cell
recording mode of the patch-clamp technique was also used to study the effect of an increase in [Ca2+]
i
on membrane currents in these cells. An increase in [Ca2+]
i
revealed two types of Ca2+-activated K+ channels, K(Ca) channels. Current through both channel types could not be observed below voltage of +80 mV with [Ca2+]
i
buffered to 100 nm or less. With patch-electrodes filled with solutions buffering [Ca2+]
i
to 10 μm both channels types could be readily observed. The activation of the first type was apparently voltage-independent since
current could be observed over the entire voltage range used from −160 to +100 mV. In addition, the current was also blocked
by charybdotoxin (CTX). The second type of K(Ca) channels in these cells could be activated with depolarizations more positive
than −40 mV from a holding potential of −80 mV. This type was blocked by CTX and paxilline. Adding paxilline to the extracellular
solution inhibited regulatory volume decrease (RVD), but could not abolish RVD. We conclude that two K(Ca) channel types exist
in human osteoblasts, an intermediate conductance K(Ca) channel and a MaxiK-like K(Ca) channel. MaxiK channels might get activated
either directly or by an increase in [Ca2+]
i
elicited through hypotonic solutions. In combination with the volume-regulated Cl− conductance in the same cells this K+ channel seems to play a vital role in volume regulation in human osteoblasts.
Received: 8 February 2000/Revised: 13 July 2000 相似文献
19.
T. Miyashita H. Tatsumi H. Furuta N. Mori M. Sokabe 《The Journal of membrane biology》2001,182(2):113-122
We identified a Ca2+-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the
patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 ± 1.3 pS (n= 8) in our standard solution. The relative ionic permeability of the channel was in the order K+= Na+ > Ca2+≫ Cl−. This channel was weakly voltage-dependent but was strongly activated by Ca2+ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca2+ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 μm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 μm), or ATP (1 mm), which increased intracellular Ca2+ concentration ([Ca2+]i), activated the channel at an estimated [Ca2+]i from 0.6 μm to 10 μm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches.
Based on this Ca2+ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing
Na+ under certain pathological conditions that will increase [Ca2+]i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells.
Received: 24 October 2000/Revised: 10 April 2001 相似文献
20.
In the present study we have studied how [Ca2+]
i
is influenced by H2O2 in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric
system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H2O2 caused a slow sustained [Ca2+]
i
increase, reaching a stable plateau after 10–15 min of perfusion. This increase induced by H2O2 was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application
of 1 mm H2O2 to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration
of CCK-8 (1 nm) or thapsigargin (0.5 μm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial
uncoupler FCCP (0.5 μm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H2O2-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and
FCCP in the presence of H2O2 did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable
calcium pools, but was followed by a sustained increase of [Ca2+]
i
. In addition, H2O2 was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca2+]
i
induced by H2O2 was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H2O2 releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H2O2 is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases.
Received: 15 May 2000/Revised: 4 October 2000 相似文献