共查询到20条相似文献,搜索用时 31 毫秒
1.
Ulyana Lalo Oleg Palygin Seyed Rasooli-Nejad Jemma Andrew Philip G. Haydon Yuriy Pankratov 《PLoS biology》2014,12(1)
Communication between neuronal and glial cells is important for many brain functions. Astrocytes can modulate synaptic strength via Ca2+-stimulated release of various gliotransmitters, including glutamate and ATP. A physiological role of ATP release from astrocytes was suggested by its contribution to glial Ca2+-waves and purinergic modulation of neuronal activity and sleep homeostasis. The mechanisms underlying release of gliotransmitters remain uncertain, and exocytosis is the most intriguing and debated pathway. We investigated release of ATP from acutely dissociated cortical astrocytes using “sniff-cell” approach and demonstrated that release is vesicular in nature and can be triggered by elevation of intracellular Ca2+ via metabotropic and ionotropic receptors or direct UV-uncaging. The exocytosis of ATP from neocortical astrocytes occurred in the millisecond time scale contrasting with much slower nonvesicular release of gliotransmitters via Best1 and TREK-1 channels, reported recently in hippocampus. Furthermore, we discovered that elevation of cytosolic Ca2+ in cortical astrocytes triggered the release of ATP that directly activated quantal purinergic currents in the pyramidal neurons. The glia-driven burst of purinergic currents in neurons was followed by significant attenuation of both synaptic and tonic inhibition. The Ca2+-entry through the neuronal P2X purinoreceptors led to phosphorylation-dependent down-regulation of GABAA receptors. The negative purinergic modulation of postsynaptic GABA receptors was accompanied by small presynaptic enhancement of GABA release. Glia-driven purinergic modulation of inhibitory transmission was not observed in neurons when astrocytes expressed dn-SNARE to impair exocytosis. The astrocyte-driven purinergic currents and glia-driven modulation of GABA receptors were significantly reduced in the P2X4 KO mice. Our data provide a key evidence to support the physiological importance of exocytosis of ATP from astrocytes in the neocortex. 相似文献
2.
Yuko Shoji Misuzu Yamaguchi-Yamada Yoshio Yamamoto 《Histochemistry and cell biology》2010,134(1):13-22
In the sensory ganglia, neurons are devoid of synaptic contacts, and ganglion neurons surrounded by one of glial cells, satellite
cells. Recent studies suggest that neurons and satellite cells interact through neurotransmitters. In the present study, intracellular
Ca2+ ([Ca2+]i) dynamics of neurons and satellite cells from one of viscerosensory ganglia, nodose ganglion (NG), were investigated by stimulation
with glutamate and its agonist and/or the antagonist of the GABAA receptor bicuculline. In the specimens containing neurons with satellite cells, glutamate and a metabotropic glutamate receptor
(mGluR) agonist t-ACPD evoked [Ca2+]i increases in both neurons and surrounding satellite cells. Moreover, bicuculline also induced [Ca2+]i increases in neurons and satellite cells. However, in the isolated neurons, bicuculline did not cause an increase in [Ca2+]i, suggesting that satellite cells are equipped with the ability to release GABA. In the neurons associated with satellite
cells, the delay time until the onset of a response was shorter in the case of glutamate stimulation with bicuculline than
that without bicuculline (107.3 ± 93.4 vs. 231.8 ± 97.0 s, p < 0.01). Furthermore, immunoreactivities for glutamate transporter, GLAST, and GABA transporter, GAT-3, were observed in
both neurons and satellite cells of NG. In conclusion, the levels of [Ca2+]i of NG neurons and surrounding satellite cells are increased by glutamate through at least mGluRs, and endogenous GABA modulates
these responses; GABA inhibition is dependent on a close association between neurons and satellite cells. Such neuron–glia
interaction in the nodose ganglion may regulate sensory information from visceral organs. 相似文献
3.
中国科学 《中国科学:生命科学英文版》2000,43(6):655-662
Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on γ-aminobutyric
acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural
nucleus (SDCN). The results showed that: (i) NMDA suppressed GABA-and muscimol (Mus)-activated currents (Igaba and IMus), respectively in the Mg2+-free external solution containing 1 μmol/L glycine at a holding potential (V
H
) of −40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 γmol/L),
inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of Igaba; (ii) when the neurons were incubated in a Ca2+-free bath or pre-loaded with a membrane-permeable Ca2+ chelator, BAPTA AM (10 μmol/L), the inhibitory effect of NMDA on IGAba disappeared. Cd2+ (10 μmol/L) or La3+ (30 μmol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppression of Igaba by NMDA application; (iii) the suppression of IGAba by NMDA was inhibited by KN-62, a calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor. These results indicated
that the inhibition of GABA response by NMDA is Ca2+-dependent and CaMKII is involved in the process of the Ca2+-dependent inhibition. 相似文献
4.
The cellular uptake of the GABA-transaminase inhibitors gamma-vinyl GABA (GVG) and gamma-acetylenic GABA (GAG) was studied in cultured neurons and astrocytes. By the use of the individual enantiomersR- andS-GVG andR- andS-GAG it could be shown that in both cell types only theS-enantiomers could be actively transported. Comparing neurons and astrocytes only neurons exhibited a high affinity uptake system forS-GVG (K
m 78.2±20.3 M;V
max 0.71±0.06 nmol · min–1 · mg–1 cell protein). In case ofS-GAG it could not be established with certainty whether the neuronal uptake was of the high affinity type. Both GVG and GAG were studied as inhibitors of GABA uptake into neurons and astrocytes.S-GVG andS-GAG were found to be weak inhibitors of GABA uptake suggesting thatS-GVG is not transported by the GABA carrier in neurons. The finding of a much more efficient uptake ofS-GVG into neurons than into astrocytes is in line with the previous observation that neuronal GABA-T is more sensitive than astrocytic GABA-T toS-GVG. 相似文献
5.
6.
The anti-SRBC response of normal, syngeneic splenocytes in the presence of cells from various tumors (Moloney leukemia spleen cells and methylcholanthrene-induced rhabdomyosarcoma cells (MC)) was tested in vitro in different culture media: RPMI 1640, BME with Hanks balanced salt solution (MEM), and CMRL 1066. The tumor-associated cells expressed an immunosuppressive effect, the degree of which varied with the culture medium used. Whereas spleen cells cultured in RPMI in the presence of tumor-associated cells were highly inhibited in their response to SRBC, those cultured in MEM were not. A full 5 to 10 times more tumor cells were required to achieve the same degree of immunosuppression in MEM. There appeared to be a correlation between the degree of immunosuppression obtained and the Ca2+ concentration of the medium. Thus the immunosuppressive effect of tumor-associated cells was greatest in cultures with RPMI 1640 (0.4 mM Ca2+), lesser in MEM (1.27 mM Ca2+), and least in CMRL 1066 (1.8 mM Ca2+). Furthermore, if the Ca2+ content of RPMI 1640 was increased to 1.4 mM Ca2+ by the addition of CaCl2, the percent suppression to the anti-SRBC response in vitro mediated by the addition of tumor cells decreased to the level found in MEM. Increasing the Mg2+ content of RPMI had no effect on tumor-mediated immunosuppression. Tumor cell replication and RNA synthesis were comparable in all media tested, regardless of Ca2+ concentration. In view of the increasing evidence for a role for Ca2+ in lymphocyte activation, we postulate herein that the Ca2+ content of the medium plays a role in the manifestation of immunosuppression by tumorassociated cells in vitro. 相似文献
7.
Ubaldo García Carlos Onetti René Valdiosera Hugo Aréchiga 《Cellular and molecular neurobiology》1994,14(1):71-88
Summary 1. Intracellular and voltage-clamp recordings were obtained from a selected population of neuroscretory (ns) cells in the X organ of the crayfish isolated eyestalk. Pulses of -aminobutyric acid (GABA) elicited depolarizing responses and bursts of action potentials in a dose-dependent manner. These effects were blocked by picrotoxin (50 µM) but not by bicuculline. Picrotoxin also suppressed spontaneous synaptic activity.2. The responses to GABA were abolished by severing the neurite of X organ cells, at about 150 µm from the cell body. Responses were larger when the application was made at the neuropil level.3. Topical application of Cd2+ (2 mM), while suppressing synaptic activity, was incapable of affecting the responses to GABA.4. Under whole-cell voltage-clamp, GABA elicited an inward current with a reversal potential dependent on the chloride equilibrium potential. The GABA effect was accompanied by an input resistance reduction up to 33% at a –50 mV holding potential. No effect of GABA was detected on potassium, calcium, and sodium currents present in X organ cells.5. The effect of GABA on steady-state currents was dependent on the intracellular calcium concentration. At 10–6
M [Ca2+]i, GABA (50 µM) increased the membrane conductance more than threefold and shifted the zero-current potential from–25 to–10 mV. At 10–9
M [Ca2+]i, GABA induced only a 1.3-fold increase in membrane conductance, without shifting the zero-current potential.6. These results support the notion that in the population of X organ cells sampled in this study, GABA acts as an excitatory neurotransmitter, opening chloride channels. 相似文献
8.
Robert S. Lasher 《Developmental neurobiology》1975,6(6):597-608
A study was made of the time course and kinetics of [3H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [3H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent Km values for GABA neurons for high and low affinity uptake were 0.33 × 10−6 M and 41.8 × 10−4 M, respectively. For nonneuronal cells, the apparent Km for high affinity uptake was 0.29 × 10−6 M. The apparent Vmax values for GABA neurons for high and low affinity uptake were 28.7 × 10−6 mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent Vmax for high affinity uptake was 0.06 × 10−6 mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 × 10−9 M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [3H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75–85%) of the [3H]GABA (4.4 × 10−6 M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent. 相似文献
9.
Removal of extracellular Ca2+ activates ion channels in the plasma membrane of defolliculated oocytes of the South Africa clawed toad Xenopus laevis. At present, there is controversy about the nature of the Ca2+-inactivated ion channels. Recently, we identified one of these channels as a Ca2+-inactivated Cl− channel (CaIC) using single channel analysis. In this work we confirm and extend previous observations on the CaIC by presenting
a decisive extension of the regulation and inhibition profile. CaIC current is reversibly blocked by the divalent and trivalent
cations Zn2+ (half-maximal blocker concentration, K1/2= 8 μm), Cu2+ (K1/2= 120 μm) and Gd3+ (K1/2= 20 μm). Furthermore, CaIC is inhibited by the specific Cl− channel blocker NPPB (K1/2≈ 3 μm). Interestingly, CaIC-mediated currents are further sensitive to the cation channel inhibitor amiloride (500 μm) but insensitive to its high affinity analogue benzamil (100 μm). An investigation of the pH-dependence of the CaIC revealed a reduction of currents in the acidic range. Using simultaneous
measurements of membrane current (I
m
), conductance (G
m
) and capacitance (C
m
) we demonstrate that Ca2+ removal leads to instant activation of CaIC already present in the plasma membrane. Since C
m
remains constant upon Ca2+ depletion while I
m
and G
m
increase drastically, no exocytotic transport of CaIC from intracellular pools and functional insertion into the plasma membrane
is involved in the large CaIC currents. A detailed overview of applicable blockers is given. These blockers are useful when
oocytes are utilized as an expression system for foreign proteins whose investigations require Ca2+-free solutions and disturbances by CaIC currents are unwanted. We further compare and discuss our results with data of Ca2+-inactivated cation channels reported by other groups.
Received: 18 June 1999/Revised: 13 August 1999 相似文献
10.
Passive Glial Cells, Fact or Artifact? 总被引:3,自引:0,他引:3
Astrocytes that are recorded in acute tissue slices of rat hippocampus using whole-cell patch-clamp, commonly exhibit voltage-activated
Na+ and K+ currents. Some reports have described astrocytes that appear to lack voltage-activated currents and proposed that these cells
constitute a subpopulation of electrophysiologically passive astrocytes. We show here that these cells can spontaneously change
during a recording unmasking expression of previously suppressed voltage-activated currents, suggesting that such cells do
not represent a subpopulation of passive astrocytes. Superfusion of a low Ca2+/EGTA solution was able to reversibly suppress voltage-activated K+ currents in cultured astrocytes. Currents were restored upon addition of normal bath Ca2+. These effects of Ca2+ on both outward and inward K+ currents were dose- and time-dependent, with increasing concentrations of Ca2+ (from 0 to 800 μm) leading to a gradual unmasking of voltage-dependent outward and inward K+ currents. The transition from an apparently passive cell to one exhibiting prominent voltage-activated currents was not associated
with any changes in membrane capacitance or access resistance. By contrast, in cells in which low access resistance or poor
seal accounted for the absence of voltage-activated currents, improvement of cell access was always accompanied by changes
in series resistance and membrane capacitance.
We propose that spillage of pipette solution containing low Ca2+/EGTA during cell approach in slice recordings and/or poor cell access, lead to a transient masking of voltage-activated currents
even in astrocytes that express prominent voltage-activated currents. These cells, however, do not constitute a subpopulation
of electrophysiologically passive astrocytes.
Received: 22 April 1998/Revised: 8 September 1998 相似文献
11.
Recent Ca2+ imaging studies in cell culture and in situ have shown that Ca2+ elevations in astrocytes stimulate glutamate release and increase neuronal Ca2+ levels, and that this astrocyte‐neuron signaling can be stimulated by prostaglandin E2 (PGE2). We investigated the electrophysiological consequences of the PGE2‐mediated astrocyte‐neuron signaling using whole‐cell recordings on cultured rat hippocampal cells. Focal application of PGE2 to astrocytes evoked a Ca2+ elevation in the stimulated cell by mobilizing internal Ca2+ stores, which further propagated as a Ca2+ wave to neighboring astrocytes. Whole‐cell recordings from neurons revealed that PGE2 evoked a slow inward current in neurons adjacent to astrocytes. This neuronal response required the presence of an astrocyte Ca2+ wave and was mediated through both N‐methyl‐D ‐aspartate (NMDA) and non‐NMDA glutamate receptors. Taken together with previous studies, these data demonstrate that PGE2‐evoked Ca2+ elevations in astrocyte cause the release of glutamate which activates neuronal ionotropic receptors. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 221–229, 1999 相似文献
12.
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
13.
Sheng-Nan Wu Hsien-Ching Huang Chia-Chen Yeh Wei-Hsin Yang Yi-Ching Lo 《Biochemical and biophysical research communications》2011,405(3):508
The membrane electroporation-induced inward current (IMEP) in pituitary tumor (GH3) cells was characterized. This current emerges irregularly when membrane hyperpolarizations to −200 mV with a holding potential of −80 mV were elicited. Neither E-4031 (10 μM), glibenclamide (30 μM), nor ZD7288 (30 μM) caused any effects on IMEP. The single-channel conductance and pore radius were estimated to be around 1.12 nS and 1.7 nm, respectively. LaCl3- and memantidine (MEM)-induced block of this current was also examined. The IC50 value for LaCl3- and MEM-induced inhibition of IMEP was 35 and 75 μM, respectively. However, unlike LaCl3, MEM (300 μM) did not exert any effect on voltage-gated Ca2+ current. In inside-out configuration, MEM applied to either external or internal surface of the excised patch did not suppress the activity of ATP-sensitive K+ channels expressed in GH3 cells, although glibenclamide significantly suppressed channel activity. This study provides the first evidence to show that MEM, a non-competitive antagonist of N-methyl D-aspartate receptors, directly inhibits the amplitude of IMEP in pituitary GH3 cells. MEM-mediated block of IMEP in these cells is unlinked to its inhibition of glutamate-induced currents or ATP-sensitive K+ currents. The channel-suppressing properties of MEM might contribute to the underlying mechanisms by which it and its structurally related compounds affect neuronal or neuroendocrine function. 相似文献
14.
High-affinity uptake of [3H]-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V
max) and, to a smaller extent, theK
m of [3H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [3H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [3H]GABA, [3H]ACHC, or [3H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution. 相似文献
15.
Pedersen MG 《Biophysical journal》2011,101(4):793-802
Secretion from many endocrine cells is a result of calcium-regulated exocytosis due to Ca2+ influx. Using the patch-clamp technique, voltage pulses can be applied to the cells to open Ca2+ channels, resulting in a measurable Ca2+ current, and evoke exocytosis, which can be seen as an increase in membrane capacitance. A common tool for evaluating the relation between Ca2+ influx and exocytosis is to plot the increase in capacitance (ΔCm) as a function of the integral of the measured Ca2+ current (Q). When depolarizations of different lengths are imposed, the rate of exocytosis is typically higher for shorter than for longer pulses, which has been suggested to result from depletion of a granule pool or from Ca2+ current inactivation. It is here demonstrated that ΔCm as a function of Q can reveal whether Ca2+ current inactivation masquerades as pool depletion. Moreover, it is shown that a convex, cooperativity-like, relation between ΔCm and Q surprisingly cannot occur as a result of cooperative effects, but can result from delays in the exocytotic process or in Ca2+ dynamics. An overview of expected ΔCm-versus-Q relations for a range of explicit situations is given, which should help in the interpretation of data of depolarization-evoked exocytosis in endocrine cells. 相似文献
16.
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
17.
Sonya Anderson Tom Brismar Elisabeth Hansson 《Cellular and molecular neurobiology》1995,15(4):439-450
Summary 1. The purpose of this study was (a) to identify if astrocytes show a similar non-Nernstian depolarization in low K+ or low Ca2+ solutions as previously found in human glial and glioma cells, and (b) to analyze the influence of the K+ conductance on the membrane potential of astrocytes.2. The membrane potential (Em) and the ionic conductance were studied with whole-cell patch-clamp technique in neonatal rat astrocytes (5–9 days in culture) and in human glioma cells (U-251MG).3. In 3.0 mM K+, Em was –75 ± 1.0 mV (mean ± SEM,n=39) in rat astrocytes and –79 ± 0.7 mV (n=5) in U-251MG cells. In both cell types Em changed linearly to the logarithm of [K+]0 between 3.0 and 160 mM K+. K+ free medium caused astrocytes to hyperpolarize to –93 ± 2.7 mV (n=21) and U-251MG cells to depolarize to –27 ± 2.1 mV (n=3).4. The I-E curve did not show inward rectification in astrocytes at this developmental stage. The slope conductance (g) exhibited only a small decrease (–19%) in K+ free solution and no significant change in 160 mM K+.5. Ba2+ (1.0 mM) depolarized astrocytes to –45 ± 2.9 mV (n=11), decreasing the slope conductance (g) by 42.4 ± 8.3% (n=11). Ca2+ free solution depolarized astrocytes to –53 ± 3.4 mV (n=12) and resulted in a positive shift of the I-E curve, increasing g by 15.3 ± 8.2% (n=8).6. Calculations indicated that a block of K+ channels explains the depolarizing effect of Ba2+. The effects of K+ free or Ca2+ free solutions on Em can be explained by a transformation of K+ channels to non-specific leakage channels. That astrocytes show a different reaction to low K+ than glioma cells can be related to the lack of inwardly rectifying K+ channels in astrocytes at this developmental stage. 相似文献
18.
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
19.
We have characterized a Ca2+-dependent Cl− current (ClCa) in cultured Sertoli cells from immature rat testis by using the whole cell recording patch-clamp technique. Cells dialyzed
with pipette solutions containing 3 mm adenoside-triphosphate (ATP) and 1 μm free Ca2+, exhibited outward currents which were inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic
acid (9-AC) but insensitive to tetraethylammonium (TEA). Dialysis of cells with pipette solutions containing less than 1 nm free Ca2+ strongly reduced the currents indicating that they were Ca2+ dependent. With cells dialyzed with Cs+ glutamate-rich pipette solutions containing 0.2 mm EGTA, 10 μm ionomycin induced outward currents having properties of Ca2+-activated Cl− currents.
With ATP-free pipette solution, the magnitude of currents was not modified suggesting the direct control by Ca2+. By contrast, addition of 0.1 mm cAMP in the pipette solution or the superfusion of cells by a permeant analogue of cAMP strongly reduced the currents. These
results may suggest that ClCa is inhibited by cAMP-dependent protein kinase.
Finally, our results do not agree with the model of primary fluid secretion by exocrine cells, but are in agreement with a
hyperpolarizing effect of cAMP in primary culture of Sertoli cells and the release of a low Cl− and bicarbonate-rich primary fluid by these cells.
Received: 30 November 1998/Revised: 2 March 1999 相似文献