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1.
Muscarinic m3 receptor-mediated changes in cytosolic Ca2+ concentration ([Ca2+]l) occur by activation of Ca2+ release channels present in the endoplasmic reticulum membrane and Ca2+ entry pathways across the plasma membrane. In this report we demonstrate the coupling of m3 muscarinic receptors to the activation of a voltage-insensitive, cation-selective channel of low conductance (3.2 ± 0.6 pS; 25 mm Ca2+ as charge carrier) in a fibroblast cell line expressing m3 muscarinic receptor clone (A9m3 cells). Carbachol (CCh)-induced activation of the cation-selective channel occurred both in whole cell and excised membrane patches (CCh on the external side), suggesting that the underlying mechanism involves receptor-channel coupling independent of intracellular messengers. In excised inside-out membrane patches from nonstimulated A9m3 cells GTP (10 μm) and GDP (10 μm) activated cation-selective channels with conductances of approximately 4.3 and 3.3 pS, (25 mm Ca2+ as charge carrier) respectively. In contrast, ATP (10 μm), UTP (10 μm) or CTP (10 μm) failed to activate the channel. Taken together, these results suggest that carbachol and guanine nucleotides regulate the activation of a cation channel that conducts calcium. Received: 14 November 1996/Revised: 4 April 1997  相似文献   

2.
A voltage-activated Ca++ channel has been identified in the apical membranes of cultured rabbit proximal tubule cells using the patch-clamp technique. With 105 mm CaCl2 solution in the pipette and 180 NaAsp in the bath, the channel had a conductance of 10.4 ± 1.0 pS (n= 8) in on-cell patches, and 9.8 ± 1.1 pS (n= 8) in inside-out patches. In both on-cell and inside-out patches, the channel is active by membrane depolarization. For this channel, the permeation to Ba++ and Ca++ is highly selective over Na+ and K+ (PCa(Ba):PNa(K) >200:1). The sensitivity to dihydropyridines is similar to that for L-type channels where the channel was blocked by nifedipine (10 μm), and activated by Bay K 8644 (5 μm). When activated by Bay K 8644, the channel showed subconductance levels. Treatment with forskolin (12.5 μm), phorbol ester (1 μm), or stretching (40 cm water) did not activate this channel. These results indicate that this Ca++ channel is mostly regulated by membrane voltage, and appears to be an epithelial class of L-type Ca++ channel. As such, it may participate in calcium reabsorption during periods of enhanced sodium reabsorption, or calcium signaling in volume regulation, where membrane depolarization occurs for prolonged periods. Received: 1 April 1996/Revised: 5 August 1996  相似文献   

3.
A large conductance, Ca2+-activated K+ channel of the BK type was examined in cultured pituitary melanotrophs obtained from adult male rats. In cell-attached recordings the slope conductance for the BK channel was ≈190 pS and the probability (P o ) of finding the channel in the open state at the resting membrane potential was low (<<0.1). Channels in inside-out patches and in symmetrical 150 mm K+ had a conductance of ≈260 pS. The lower conductance in the cell-attached recordings is provisionally attributed to an intracellular K+ concentration of ≈113 mm. The permeability sequence, relative to K+, was K+ > Rb+ (0.87) > NH+ 4 (0.17) > Cs+≥ Na+ (≤0.02). The slope conductance for Rb+ was much less than for K+. Neither Na+ nor Cs+ carried measurable currents and 150 mm internal Cs+ caused a flickery block of the channel. Internal tetraethylammonium ions (TEA+) produced a fast block for which the dissociation constant at 0 mV (K D (0 mV)) was 50 mm. The K D (0 mV) for external TEA+ was much lower, 0.25 mm, and the blocking reaction was slower as evidenced by flickery open channel currents. With both internal and external TEA+ the blocking reaction was bimolecular and weakly voltage dependent. External charybdotoxin (40 nm) caused a large and reversible decrease of P o . The P o was increased by depolarization and/or by increasing the concentration of internal Ca2+. In 0.1 μm Ca2+ the half-maximal P o occurred at ≈100 mV; increasing Ca2+ to 1 μm shifted the voltage for the half-maximal P o to −75 mV. The Ca2+ dependence of the gating was approximated by a fourth power relationship suggesting the presence of four Ca2+ binding sites on the BK channel. Received: 23 October/Revised: 15 December 1995  相似文献   

4.
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   

5.
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   

6.
Large Conductance Ca2+-Activated K+ Channels in Human Meningioma Cells   总被引:2,自引:0,他引:2  
Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures. In whole cells, depolarization to voltages higher than +80 mV evoked a large K+ outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC50= 5.3 mm). Raising the internal Ca2+ from 10 nm to 2 mm shifted the voltage of half-maximum activation (V 1/2) of the K+ current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca2+-activated (BK Ca ) K+ channel with a conductance of 296 pS (130 mm K+ at both sides of the patch). V 1/2 of single-channel currents was +6, −12, −46, and −68 mV in the presence of 1, 10, 100, and 1000 μm Ca2+, respectively, at the internal face of the patch. In cell-attached patches the open probability (P o ) of BK Ca channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K+ currents with 10 nm Ca2+ in the pipette. Application of 20 μm cytochalasin D increased P o of BK Ca channels in cell-attached patches within minutes. These data suggest that the activation of BK Ca channels in meningioma cells does not only depend on voltage and internal Ca2+ but is also controlled by the cytoskeleton. Received 18 June 1999/Revised: 18 January 2000  相似文献   

7.
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   

8.
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   

9.
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   

10.
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   

11.
The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K Ca ) channel and (iii) a small K+ channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear current-voltage (I-V) relationship (27 pS) and was equally conductive for Na+ and K+, but not permeable to Cl or N-methyl-d-glucamine. Channel activity required the presence of Ca2+ at the cytosolic face, but was detected at Ca2+ concentrations as low as 10−7 m (open probability (P o ) = 0.11 ± 0.03, n= 8). Gadolinium decreased P o of the NSC channel from both the external and cytosolic side (IC50∼ 0.6 μm). NSC currents were decreased by amiloride (10 μm− 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K+ could be fitted to the I-V relationship in asymmetrical K+ and Na+ solutions. The channel was impermeable to Cl and N-methyl-d-glucamine. P o of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca2+. TEA (20 mm), charybdotoxin (100 nm) and Ba2+ (1 mm) but not amiloride (1 mm) reduced P o from the extracellular side. In contrast, external flufenamic acid (100 μm) increased P o and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward current. We conclude that the NSC channel is Ca2+ activated, voltage-insensitive and involved in both constitutive K+ and Na+ reabsorption from endolymph while the BK channel might participate in the K+ pathway under stimulated conditions that produce an elevated intracellular Ca2+ or depolarized membrane potential. Received: 14 October 1999/Revised: 10 December 1999  相似文献   

12.
Melanoma cells are transformed melanocytes of neural crest origin. K+ channel blockers have been reported to inhibit melanoma cell proliferation. We used whole-cell recording to characterize ion channels in four different human melanoma cell lines (C8161, C832C, C8146, and SK28). Protocols were used to identify voltage-gated (KV), Ca2+-activated (KCa), and inwardly rectifying (KIR) K+ channels; swelling-sensitive Cl channels (Clswell); voltage-gated Ca2+ channels (CaV) and Ca2+ channels activated by depletion of intracellular Ca2+ stores (CRAC); and voltage-gated Na+ channels (NaV). The presence of Ca2+ channels activated by intracellular store depletion was further tested using thapsigargin to elicit a rise in [Ca2+] i . The expression of K+ channels varied widely between different cell lines and was also influenced by culture conditions. KIR channels were found in all cell lines, but with varying abundance. Whole-cell conductance levels for KIR differed between C8161 (100 pS/pF) and SK28 (360 pS/pF). KCa channels in C8161 cells were blocked by 10 nm apamin, but were unaffected by charybdotoxin (CTX). KCa channels in C8146 and SK28 cells were sensitive to CTX (K d = 4 nm), but were unaffected by apamin. KV channels, found only in C8146 cells, activated at ∼−20 mV and showed use dependence. All melanoma lines tested expressed CRAC channels and a novel Clswell channel. Clswell current developed at 30 pS/sec when the cells were bathed in 80% Ringer solution, and was strongly outwardly rectifying (4:1 in symmetrical Cl). We conclude that different melanoma cell lines express a diversity of ion channel types. Received: 2 April 1996/Revised: 22 August 1996  相似文献   

13.
These experiments were conducted to determine the membrane K+ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K+ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca2+-dependent outward K+ current, 0.57 ± 0.13 nS/pF at −70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca2+-activated, voltage-dependent K+ channel (KCa) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the KCa activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K+ channel, 21 ± 1 pS at positive transmembrane potential (V m ) and 145 ± 13 pS at negative V m . Glibenclamide (50 μm), Ba2+ (1 mm) and quinine (100 μm) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K+ channels in inside/out patches in a dose-dependent manner, and the IC50= 46 μm. The identity of this K+ channel with an ATP-sensitive K+ channel (KATP) was confirmed by its inhibition with ATP (2 mm) and by its activation with diazoxide (100 μm). We conclude that plasma membranes of HTB-9 cells contain the KCa and a lower conductance K+ channel with properties consistent with a sulfonylurea receptor-linked KATP. Received: 12 June 1997/Revised: 21 October 1997  相似文献   

14.
An amiloride-sensitive, Ca2+-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role in stimulation of Na+ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca2+ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium, terbutaline, a beta agonist, increased the open probability (P o ) of the NSC channel to 0.62 ± 0.07 from 0.03 ± 0.01 (mean ±se; n= 8) 30 min after application of terbutaline in a solution containing 1 mm Ca2+. The P o of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca2+ to 0.26 ± 0.05 (n= 8). The cytosolic Ca2+ concentration ([Ca2+] c ) in the presence and absence of extracellular Ca2+ was, respectively, 100 ± 6 and 20 ± 2 nm (n= 7) 30 min after application of terbutaline. The cytosolic Cl concentration ([Cl] c ) in the presence and absence of extracellular Ca2+ was, respectively, 20 ± 1 and 40 ± 2 mm (n= 7) 30 min after application of terbutaline. The diminution of [Ca2+] c from 100 to 20 nm itself had no significant effects on the P o if the [Cl] c was reduced to 20 mm; the P o was 0.58 ± 0.10 at 100 nm [Ca2+] c and 0.55 ± 0.09 at 20 nm [Ca2+] c (n= 8) with 20 mm [Cl] c in inside-out patches. On the other hand, the P o (0.28 ± 0.10) at 20 nm [Ca2+] c with 40 mm [Cl] c was significantly lower than that (0.58 ± 0.10; P < 0.01; n= 8) at 100 nm [Ca2+] c with 20 mm [Cl] c , suggesting that reduction of [Cl] c is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca2+ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl] c . Received: 11 August 2000/Revised: 4 December 2000  相似文献   

15.
Properties of large conductance Ca2+-activated K+ channels were studied in the soma of motoneurones visually identified in thin slices of neonatal rat spinal cord. The channels had a conductance of 82 ± 5 pS in external Ringer solution (5.6 mm K+ o //155 mm K+ i ) and 231 ± 4 pS in external high-K o solution (155 mm K+ o //155 mm K+ i ). The channels were activated by depolarization and by an increase in internal Ca2+ concentration. Potentials of half-maximum channel activation (E50) were −13, −34, −64 and −85 mV in the presence of 10−6, 10−5, 10−4 and 10−3 m internal Ca2+, respectively. Using an internal solution containing 10−4 m Ca2+, averaged KCa currents showed fast activation within 2–3 msec after a voltage step to +50 mV. Averaged KCa currents did not inactivate during 400 msec voltage pulses. External TEA reduced the apparent single-channel amplitude with a 50% blocking concentration (IC50) of 0.17 ± 0.02 mm. KCa channels were completely suppressed by externally applied 100 mm charybdotoxin. It is concluded that KCa channels activated by Ca2+ entry during the action potential play an important role in the excitability of motoneurones. Received: 7 November 1996/Revised: 29 October 1997  相似文献   

16.
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   

17.
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   

18.
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   

19.
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   

20.
Single channel and [3H]ryanodine binding measurements were performed to test for a direct functional interaction between 2,3-butanedione 2-monoxime (BDM) and the skeletal and cardiac muscle sarcoplasmic reticulum Ca2+ release channels (ryanodine receptors). Single channel measurements were carried out in symmetric 0.25 m KCl media using the planar lipid bilayer method. BDM (1–10 mm) activated suboptimally Ca2+-activated (0.5–1 μm free Ca2+) single, purified and native cardiac and skeletal release channels in a concentration-dependent manner by increasing the number of channel events without a change of single channel conductances. BDM activated the two channel isoforms when added to either side of the bilayer. At a maximally activating cytosolic Ca2+ concentration of 20 μm, BDM was without effect on the cardiac channel, whereas it inhibited skeletal channel activities with IC50≈ 2.5 mm. In agreement with single channel measurements, high-affinity [3H]ryanodine binding to the two channel isoforms was increased in a concentration-dependent manner at ≤1 μm Ca2+. BDM was without a noticeable effect at low (≤0.01 μm) Ca2+ concentrations. At 20 μm Ca2+, BDM inhibited the skeletal but not cardiac channel. These results suggest that BDM regulates the Ca2+ release channels from the sarcoplasmic reticulum of skeletal and cardiac muscle in a concentration, Ca2+ and tissue-dependent manner. Received: 31 December 1998/Revised: 9 March 1999  相似文献   

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