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1.
Luis Gandía Baldomero Lara Julita S. Imperial Mercedes Villarroya Almudena Albillos Rosario Maroto A. G. García Baldomero M. Olivera 《Pflügers Archiv : European journal of physiology》1997,435(1):55-64
The characteristics of the binding sites for the Conus magus toxins ω-conotoxin MVIIC and ω-conotoxin MVIID, as well as their effects on K+-evoked 45Ca2+ entry and whole-cell Ba2+ currents (I
Ba), and K+-evoked catecholamine secretion have been studied in bovine adrenal chromaffin cells. Binding of [125I] ω-conotoxin GVIA to bovine adrenal medullary membranes was displaced by ω-conotoxins GVIA, MVIIC and MVIID with IC50 values of around 0.1, 4 and 100 nM, respectively. The reverse was true for the binding of [125I] ω-conotoxin MVIIC, which was displaced by ω-conotoxins MVIIC, MVIID and GVIA with IC50 values of around 30, 80 and 1.200 nM, respectively. The sites recognized by ω-conotoxins MVIIC and MVIID in bovine brain
exhibited higher affinities (IC50 values of around 1 nM). Both ω-conotoxin MVIIC and MVIID blocked I
Ba by 70–80%; the higher the [Ba2+]o of the extracellular solution the lower the blockade induced by ω-conotoxin MVIIC. This was not the case for ω-conotoxin
MVIID; high Ba2+ (10 mM) slowed down the development of blockade but the maximum blockade achieved was similar to that obtained in 2 mM Ba2+. A further difference between the two toxins concerns their reversibility; washout of ω-conotoxin MVIIC did not reverse the
blockade of I
Ba while in the case of ω-conotoxin MVIID a partial, quick recovery of current was produced. This component was irreversibly
blocked by ω-conotoxin GVIA, suggesting that it is associated with N-type Ca2+ channels. Blockade of K+-evoked 45Ca2+ entry produced results which paralleled those obtained by measuring I
Ba. Thus, 1 μM of each of ω-conotoxin GVIA and MVIIA inhibited Ca2+ uptake by 25%, while 1 μM of each of ω-conotoxin MVIIC and MVIID caused a 70% blockade. K+-evoked catecholamine secretory responses were not reduced by ω-conotoxin GVIA (1 μM). In contrast, at 1 μM both ω-conotoxin
MVIIC and MVIID reduced the exocytotic response by 70%. These data strengthen the previously established conclusion that Q-type
Ca2+ channels that contribute to the regulation of secretion and are sensitive to ω-conotoxins MVIIC and MVIID are present in
bovine chromaffin cells. These channels, however, seem to possess binding sites for ω-conotoxins MVIIC and MVIID whose characteristics
differ considerably from those described to occur in the brain; they might represent a subset of Q-type Ca2+ channels or an entirely new subtype of voltage-dependent high-threshold Ca2+ channel.
Received: 16 April 1997 / Received after revision: 10 July 1997 / Accepted: 23 July 1997 相似文献
2.
Manuela G. López Almudena Albillos María Teresa de la Fuente Ricardo Borges Luis Gandía Emilio Carbone Antonio G. García Antonio R. Artalejo 《Pflügers Archiv : European journal of physiology》1994,427(3-4):348-354
Depolarizing 1-s pulses to 0 mV from a holding potential of −70 mV, induced whole-cell currents through Ca2+ channels (I
Ca) in patch-clamped cat adrenal medulla chromaffin cells. The dihydropyridine (DHP) furnidipine (3 μM) reduced the peak current
by 47% and the late current by 80%. ω-Conotoxin GVIA (CgTx, 1 μM) reduced the peak I
Ca by 42% and the late I
Ca by 55%. Pulses (10 s duration) with 70 mM K+/2.5 mM Ca2+ solution (70 K+/2.5 Ca2+), applied to single fura-2-loaded cat chromaffin cells increased the cytosolic Ca2+ concentration ([Ca2+]i from 0.1 to 2.21 μM; this increase was reduced by 43.7% by furnidipine and by 42.5% by CgTx. In the perfused cat adrenal
gland, secretion evoked by 10-s pulses of 70 K+/2.5 Ca2+ was reduced by 25% by CgTx and by 96% by furnidipine. Similar results were obtained when secretion from superfused isolated
cat adrenal chromaffin cells was studied and when using a tenfold lower [Ca2+]o. The results are compatible with the existence of DHP-sensitive (L-type) as well as CgTx-sensitive (N-type) voltage-dependent
Ca2+ channels in cat chromaffin cells. It seems, howevever, that though extracellular Ca2+ entry through both channel types leads to similar increments of averaged [Ca2+]i, the control of catecholamine release is dominated only by Ca2+ entering through L-type Ca2+ channels. This supports the idea of a preferential segregation of L-type Ca2+ channels to localized “hot spots” in the plasmalemma of chromaffin cells where exocytosis occurs. 相似文献
3.
Jesús-Miguel Hernández-Guijo Luis Gandía Baldomero Lara A. G. García 《Pflügers Archiv : European journal of physiology》1998,437(1):104-113
Applying 10-s pulses of 10 mM Ba2+ to resting or K+-depolarized (70 mM) bovine adrenal chromaffin cells superfused with a nominal 0Ca2+ solution produced a large catecholamine secretory peak. In contrast, pulses of 10 mM Sr2+ or Ca2+ did not induce secretion from polarized resting cells, and induced smaller and narrower secretory peaks from depolarized
cells; the areas of the secretory peaks from depolarized cells were 1.87, 3.06 and 27.4 nA s, respectively, for Ca2+, Sr2+ and Ba2+. Ca2+ channel currents in isolated cells or in cells surrounded by other unpatched cells (cell cluster) were studied with either
the continuous-flow or the flow-stop method. When applied to an isolated cell, flow-stop reduced the amplitude of I
Ca by 19%, I
Sr by 31%, and I
Ba by 53%, compared with the current amplitude measured under continuous-flow conditions. This decrease in current amplitude
was accompanied by a pronounced slowing down of current activation and could be largely relieved by applying strong depolarizing
prepulses (facilitation). Under continuous-flow conditions, 10 μM exogenous ATP reduced (about 50%) I
Ca, I
Sr and I
Ba similarly. On the other hand, the use of Na+ as a charge carrier through Ca2+ channels, or intracellular dialysis with 1 mM BAPTA prevented the modulation of current by flow-stop. In cell clusters, activating
secretion from unpatched cells, by either 10 mM Ba2+, 100 μM acetylcholine or 70 mM K+, caused a pronounced slowing down of current activation, as well as a decrease of its magnitude in the voltage-clamped cell
immersed in the cluster. Such modulation of isolated cells was not observed. These data are compatible with the idea that
the secretory activity of adrenal medullary chromaffin cells ”in situ” controls the activity of their Ca2+ channels through autocrine/paracrine mechanisms.
Received: 29 June 1998 / Received after revision: 20 August 1998 / Accepted: 1 September 1998 相似文献
4.
Calcium channel subtypes in porcine adrenal chromaffin cells 总被引:3,自引:0,他引:3
Naoki Kitamura Toshio Ohta Shigeo Ito Y. Nakazato 《Pflügers Archiv : European journal of physiology》1997,434(2):179-187
The effects of nifedipine, ω-conotoxin GVIA (ω-CgTx) and ω-agatoxin IVA (ω-AgTx) on Ca2+ currents, a 60-mM-K+-induced increase in intracellular Ca2+ concentration ([Ca2+]i) and catecholamine secretion were examined to clarify the subtypes of Ca2+ channels in cultured adrenal chromaffin cells from the pig. Nifedipine, ω-CgTx, and ω-AgTx inhibited Ca2+ currents in a dose-dependent manner, suggesting the presence of L-, N- and P-type Ca2+ channels. The maximal doses of nifedipine (10 μM), ω-CgTx (1 μM), and ω-AgTx (0.1 μM) inhibited Ca2+ currents to 85%, 22%, and 94% of control currents, respectively. The inhibitory effects of these three blockers were observed
in the same cell, indicating that at least three subtypes of Ca2+ channels are present in porcine chromaffin cells. The increase in [Ca2+]i and catecholamine secretion induced by 60 mM K+ were inhibited equally by nifedipine (10 μM) and ω-CgTx (1 μM), but not by ω-AgTx (0.1 μM). These results suggest that L-,
N- and P-type Ca2+ channels are present in porcine adrenal chromaffin cells, and that the major pathways of Ca2+ entry evoked by a high concentration of K+ are L- and N-type Ca2+ channels.
Received: 6 September 1996 / Received after revision: 3 February 1997 / Accepted: 18 February 1997 相似文献
5.
Almudena Albillos Antonio G. García Baldomero Olivera Luis Gandía 《Pflügers Archiv : European journal of physiology》1996,432(6):1030-1038
This study was undertaken to reassess the set of voltage-dependent Ca2+ channel subtypes expressed by bovine adrenal chromaffin cells maintained in primary cultures. Previous views on the pharmacology
of such channels had to be revised in the light of the novel data which arose from the use in this study of low and high micromolar
concentrations of ω-agatoxin IVA, and low (2 mM) and high (10 mM) concentrations of the charge carrier Ba2+. Whole-cell Ba2+ currents (IBa) through Ca2+ channels were elicited in voltage-clamped chromaffin cells, with a holding potential of –80 mV and depolarising pulses to
0 mV. Mean peak I
Ba was 425 pA in 2 mM Ba2+ (59 cells) and 787 pA in 10 mM Ba2+ (42 cells). In 2 mM Ba2+, ω-conotoxin MVIIC (3 μM) inhibited I
Ba by 79%; in 10 mM Ba2+, the blockade developed much more slowly and reached only 44%. A low concentration of ω-agatoxin IVA (20 nM) inhibited I
Ba by 9%; 2 μM inhibited I
Ba by 60%. This blockade was similar in low and high Ba2+ concentrations. After giving furnidipine (3 μM) and ω-conotoxin GVIA (1 μM), 2 μM ω-agatoxin IVA inhibited the remaining current
(about 40–45%); this blockade was independent of the Ba2+ concentration. The current could be fully blocked by the cocktail furnidipine/ω-conotoxin GVIA/high ω-agatoxin IVA, both in
low and high Ba2+ concentrations. The large Q-type channel component of I
Ba is blocked by micromolar concentrations of ω-agatoxin IVA and ω-conotoxin MVIIC. While solutions with a high Ba2+ concentration strongly delayed the development of blockade by ω-conotoxin MVIIC, the blockade by high concentrations of ω-agatoxin
IVA was equally effective in solutions with a low or a high Ba2+ concentration. Hence, the use of appropriate Ba2+ and toxin concentrations in this study reveals that P-type Ca2+ channels are poorly expressed in bovine chromaffin cells; in contrast, a robust component of the current depends on Q-type
Ca2+ channels. An R-type residual current is not present in these cells.
Received: 22 April 1996 / Received after revision: 11 June 1990 / Accepted: 11 June 1996 相似文献
6.
Yu. V. Bukanova E. I. Solntseva V. G. Skrebitskii 《Bulletin of experimental biology and medicine》1998,126(4):1006-1009
A high-threshold (−20 mV) K+ current was recorded from isolated edible snail neurons by a two-microelectrode voltage clamp technique. This current consisted
of three components: fast-inactivating K+ currents (IA), noninactivating K+ current (IKD), and Ca2+-dependent K+ current (IK(Ca)). Different cells had one to three components of K+ current. Vinpocetine increased IA, moderately inhibited IKD (by 30–50%) and strongly suppressed IK(Ca) (by 60–90%). Inhibition of IK(Ca) was not related to the effect of vinpocetine on the inward Ca2+ current. When K+ current consisted of all three components, the effect of vinpocetine on the ionic current amplitude was opposite at different
potentials.
Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 10, pp. 408–411, October, 1998 相似文献
7.
The exposure of frog skeletal muscle to caffeine (3–4 mM) generates an increase of the K+ (42K+) efflux rate coefficient (k
K,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca2+ ([Ca2+]i), because the effect is mimicked by ionomycin (1.25 μM), a Ca2+ ionophore. Second, the inhibition of caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) by 40 μM tetracaine significantly reduced the increase in k
K,o (Δk
K,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca2+-dependent K+ channels (BKCa channels) reduced Δk
K,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca2+-dependent K+ channels (SKCa channels), did not affect Δk
K,o. Fifth, tolbutamide (800 μM), an inhibitor of KATP channels, reduced Δk
K,o by about 23%. Sixth, Ba2+, a blocker of most K+ channels, did not preclude the caffeine-induced Δk
K,o. Seventh, omitting Na+ from the external medium reduced Δk
K,o by about 40%. Eight, amiloride (5 mM) decreased Δk
K,o by 65%. It is concluded that the caffeine-induced rise of [Ca2+]i increases K+ efflux, through the activation of: (1) two channels (BKCa and KATP) and (2) an external Na+-dependent amiloride-sensitive process.
Received: 13 March 1998 / Received after revision: 17 June 1998 / Accepted: 14 September 1998 相似文献
8.
J. Ashot Kozak D. E. Logothetis 《Pflügers Archiv : European journal of physiology》1997,433(6):679-690
Ca2+-dependent conductances have been hypothesized to play a role in the bursting pattern of electrical activity of insulin-secreting
β cells in response to high plasma glucose. A Maxi K+ channel has received the most attention, while a low-conductance Ca2+-activated K+ current has also been identified. We used an increasingly popular β cell model system, the βTC-3 cell line, and the perforated-patch
technique to describe the properties of a novel Ca2+-dependent Cl–current [I
Cl(Ca)] in insulin-secreting pancreatic β cells. The reported ICl(Ca) could be activated under physiological Ca2+ concentrations and is the first of its kind to be described in pancreatic insulin-secreting cells. We found that long depolarizing
steps above –20 mV elicited an outward current which showed slow inward relaxation upon repolarization to negative membrane
potentials. Both the outward currents and the inward tails showed dependence on Ca2+ influx: their current/voltage (I/V) relations followed that of the ”L-like” Ca2+ current (I
Ca) present in these cells; they were blocked completely by the removal of external Ca2+ or application of Cd2+ at concentrations sufficient for complete block of I
Ca; and their magnitude increased with the depolarizing step duration. Moreover, the inward tail decayed monoexponentially with
a time constant which at voltages negative to activation of I
Ca showed a weak linear voltage dependence, while at voltages positive to activation of I
Ca it followed the voltage dependence of I
Ca. This Ca2+-dependent current reversed at –21.5 mV and when the external Cl–concentration was reduced from 159 mM to 62 mM the reversal potential shifted by ≈+20 mV as predicted by the Nernst relation
for a Cl–-selective current. Cl–channel blockers such as DIDS (100 μM) and niflumic acid (100 μM) blocked this current. We concluded that this current was
a Ca2+-dependent Cl–current [I
Cl(Ca)]. From substitution of the external Cl–with various monovalent anions and from the reversal potentials we obtained the following permeability sequence for I
Cl(Ca): I
–>NO3
–>Br–>Cl–>Acetate–.
Received: 10 October 1996 / Received after revision and accepted: 19 December 1996 相似文献
9.
Baldomero Lara Luis Gandía Rafael Martínez-Sierra Andrés Torres A. G. García 《Pflügers Archiv : European journal of physiology》1998,435(4):472-478
This study uses a new strategy to investigate the hypothesis that, of the various Ca2+ channels expressed by a neurosecretory cell, a given channel subtype is coupled more tightly to the exocytotic apparatus
than others. The approach is based on the prediction that the degree of inhibition of the secretory response by various Ca2+ channel blockers will differ at low (0.5 mM) and high (5 mM) extracellular Ca2+ concentrations ([Ca2+]o). So, at low [Ca2+]o the K+-evoked catecholamine release from superfused bovine chromaffin cells was depressed 60–70% by 2 μM ω-agatoxin IVA (P/Q-type
Ca2+ channel blockade), by 3 μM ω-conotoxin MVIIC (N/P/Q-type Ca2+ channel blockade), or by 3 μM lubeluzole (N/P/Q-type Ca2+ channel blockade); in high [Ca2+]o these blockers inhibited the responses by only 20–35%. At 1–3 μM ω-conotoxin GVIA (N-type Ca2+ channel blockade) or 3 μM furnidipine (L-type Ca2+ channel blockade), secretion was inhibited by 30 and 50%, respectively; such inhibitory effects were similar in low or high
[Ca2+]o. Combined furnidipine plus ω-conotoxin MVIIC, ω-agatoxin IVA or ω-conotoxin GVIA exhibited additive blocking effects at
both Ca2+ concentrations. The results suggest that Q-type Ca2+ channels are coupled more tightly to exocytotic active sites, as compared to L-type channels. This hypothesis if founded
in the fact that external Ca2+ that enters the cell through a Ca2+ channel located near to chromaffin vesicles will saturate the K+ secretory response at both [Ca2+]o, i.e. 0.5 mM and 5 mM. In contrast, Ca2+ ions entering through more distant channels will be sequestered by intracellular buffers and, thus, will not saturate the
secretory machinery at lower [Ca2+]o.
Received: 23 September 1997 / Received after revision: 29 October 1997 / Accepted: 30 October 1997 相似文献
10.
Human adrenal chromaffin cell calcium channels: drastic current facilitation in cell clusters, but not in isolated cells 总被引:6,自引:0,他引:6
Luis Gandía Inés Mayorgas Pedro Michelena Inmaculada Cuchillo Ricardo de Pascual Francisco Abad Jesús M. Novalbos Eduardo Larrañaga A. G. García 《Pflügers Archiv : European journal of physiology》1998,436(5):696-704
Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue
was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope
and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba2+ (holding potential, V
h=–80 mV) revealed the presence of only high-threshold voltage-dependent Ca2+ channels; T-type Ca2+ channels were not seen. By using supramaximal concentrations of selective Ca2+ channel blockers, the whole-cell I
Ba could be fractionated into various subcomponents. Thus, I
Ba had a 25% fraction sensitive to 1 μM nifedipine (L-type channels), 21% sensitive to 1 μM ω-conotoxin GVIA (N-type channels),
and 60% sensitive to 2 μM ω-agatoxin IVA (P/Q-type channels). The activation of I
Ba was considerably slowed down, and the peak current was inhibited upon superfusion with 10 μM ATP. The slow activation and
peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of
I
Ba was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped
cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca2+ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is
markedly increased by Ba2+. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca2+ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each).
This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel
type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted
for 5–50%. The results also provide strong support for the hypothesis that Ca2+ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines,
i.e. ATP and opiates.
Received: 1 May 1998 / Received after revision and accepted: 21 May 1998 相似文献
11.
Akinori Kuruma Masayasu Hiraoka S. Kawano 《Pflügers Archiv : European journal of physiology》1998,436(6):976-983
We investigated how Ca2+-sensitive transient outward current, I
to(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na+ (Na+
i) using the whole-cell patch-clamp technique at 36°C. In cells dialysed with Na+-free solutions,the application of nicardipine (5 μM) to block L-type Ca2+ current (I
Ca) completely inhibited I
to(Ca). In cells dialysed with a [Na+]i≥5 mM, however, I
to(Ca) could be observed after blockade of I
Ca, indicating the activity of an I
Ca-independent component. The amplitude of I
Ca-independent I
to(Ca) increased with voltage in a [Na+]i-dependent manner. The block of Ca2+ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked I
Ca-independent I
to(Ca). In Ca2+-free bath solution I
to(Ca) was completely abolished. The application of 2 mM Ni2+ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na+/Ca2+ exchange, or perfusion with pipette solution containing XIP (10 μM), a selective blocker of the exchanger, blocked I
Ca-independent I
to(Ca). From these results we conclude that, in the presence of Na+
i, I
to(Ca) can be activated via Ca2+-induced Ca2+ release triggered by Na+/Ca2+ exchange operating in the reverse mode after blockade of I
Ca.
Received: 20 January 1998 / Received after revision: 6 July 1998 / Accepted: 25 July 1998 相似文献
12.
Endothelin depolarizes myocytes from porcine coronary and human mesenteric arteries through a Ca-activated chloride current 总被引:12,自引:0,他引:12
The effect of endothelin (ET) on membrane potential and current was studied in myocytes isolated from porcine coronary or from human mesenteric arteries at 3.6 mM extracellular Ca2+ concentration and 37° C. ET (1–100 nM) induced cell shortening and membrane depolarization from a resting potential of –50 mV to about –15 mV. Ca currents (I
Ca, L-type) were transiently reduced by ET. At –50 mV, ET induced an inward current that peaked within 2 s and fell within 10 s to a sustained level. The current could be enlarged by reducing bath extracellular Cl– ion concentration, but removal of extracellular Na+ ions had no effect. The voltage dependence suggests that the ET-induced current is a Cl current (I
Cl) at potentials negative to –30 mV; at more positive potentials K currents (I
K, Ca) are superimposed. The effects of ET on I
Ca, I
Cl, I
K, Ca contraction were prevented by intracellular Ca chelators, suggesting a Ca-dependent activation mechanism. The ET effects were abolished by pretreatment with 20 mM caffeine or prior cell-dialysis with heparin [thought to block inositol triphosphate-induced sarcoplasmic reticular Ca release]. The results suggest that ET releases Ca from the SR through a phosphoinositol response and that the released Ca acts as second messenger in modulating the membrane currents. 相似文献
13.
Michael C. Sanguinetti Nancy K. Jurkiewicz 《Pflügers Archiv : European journal of physiology》1992,420(2):180-186
We sought to determine whether extracellular Ca2+ (Ca
e
2+
) and K+ (K
e
+
) play essential roles in the normal functioning of cardiac K+ channels. Reports by others have shown that removal of Ca
e
2+
and K
e
+
alters the gating properties of neural delayed rectifier (I
K) and A-type K+ currents, resulting in a loss of normal cation selectivity and voltage-dependent gating. We found that removal of Ca
e
2+
and K
e
+
from the solution bathing guinea pig ventricular myocytes often induced a leak conductance, but did not affect the ionic selectivity or time-dependent activation and deactivation properties of I
K. The effect of [K+]e on the magnitude of the two components of cardiac I
K was also examined. I
K in guinea pig myocytes is comprised of two distinct types of currents: I
Kr (rapidly activating, rectifying) and I
Ks (slowly activating). The differential effect of Ca
e
2+
on the two components of I
K (previously shown to shift the voltage dependence of activation of the two currents in opposite directions) was exploited to determine the role of K
e
+
on the magnitude of I
Ks and I
Kr. Lowering [K+]e from 4 to 0 mM increased I
Ks, as expected from the change in driving force for K+, but decreased I
Kr. The differential effect of [K+]e on the two components of cardiac I
K may explain the reported discrepancies regarding modulation of cardiac I
K conductance by this cation. 相似文献
14.
Effects of ruthenium red on membrane ionic currents in urinary bladder smooth muscle cells of the guinea-pig 总被引:2,自引:0,他引:2
Masaru Hirano Y. Imaizumi Katsuhiko Muraki A. Yamada Minoru Watanabe 《Pflügers Archiv : European journal of physiology》1998,435(5):645-653
Three major ionic currents, Ca2+-dependent K+ current (I
K-Ca), delayed rectifier type K+ current (I
kd) and Ca2+ current (I
Ca), were activated by depolarization under whole-cell clamp in single smooth muscle cells isolated from guinea-pig urinary
bladder. Externally applied ruthenium red (RuR) reduced the amplitude of I
K-Ca and I
Ca at 0 mV (IC50 values were 4.2 and 5.6 μM, respectively), but did not affect I
Kd. Spontaneous transient outward currents (STOCs) and caffeine-induced outward currents (I
caf) at –30 mV were reduced by external 10 μM RuR. When 10 μM RuR was added to the pipette solution, I
K-Ca during depolarization, STOCs and I
caf significantly decreased with time. RuR did not change the unitary current amplitude of the large-conductance Ca2+-dependent K+ (BK) channels, but reduced the open probability of the channel under excised patch-clamp recording mode. RuR reduced the
channel activity more effectively from the cytosolic face than from the other. This inhibition decreased when the cytosolic
Ca2+ concentration was increased. These results indicate that RuR blocks BK and Ca2+ channels in urinary bladder smooth muscle cells. The decrease in I
K-Ca, STOCs and I
caf by RuR is attributable to the direct inhibition of BK channel activity, probably in addition to the inhibition of Ca2+ release from storage sites. The direct inhibition of BK channel activity by RuR may be related to the interaction of RuR
with the Ca2+-binding sites of the channel protein.
Received: 15 October 1997 / Received after revision and accepted: 25 November 1997 相似文献
15.
Hidenori Sako Nicolas Sperelakis A. Yatani 《Pflügers Archiv : European journal of physiology》1998,435(5):749-752
β-adrenergic receptor (β-AR) stimulation increases cardiac L-type Ca2+ channel (CaCh) currents via cAMP-dependent phosphorylation. We report here that the affinity and maximum response of CaCh
to isoproterenol (Iso), in mouse ventricular myocytes were significantly higher when Ba2+ was used as the charge carrier (I
Ba) instead of Ca2+ (I
Ca). The EC50 and maximum increase of peak currents were 43.7 ± 7.9 nM and 1.8 ± 0.1-fold for I
Ca and 23.3 ± 4.7 nM and 2.4 ± 0.1-fold for I
Ba. When cells were dialyzed with the faster Ca2+ chelator, BAPTA, both sensitivity and maximum response of I
Ca to Iso were significantly augmented compared to cells with EGTA (EC50 of 23.1 ± 5.2 nM and maximal increase of 2.2 ± 0.1-fold). Response of I
Ca to forskolin was also significantly increased when cells were dialyzed with BAPTA or when currents were measured in Ba2+. In contrast, depletion of the sarcoplasmic reticulum (SR) Ca2+ stores by ryanodine did not alter sensitivity of I
Ca to Iso or forskolin. These results suggest that the Ca2+ entering through CaCh regulates cAMP-dependent phosphorylation, and such negative feedback may play a significant role in
cellular Ca2+ homeostasis and contraction in cardiac cells during β-AR stimulation.
Received: 10 December 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998 相似文献
16.
Y. Hayabuchi Yutaka Nakaya Suguru Matsuoka Yasuhiro Kuroda 《Pflügers Archiv : European journal of physiology》1998,436(4):509-514
Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca2+-activated K+ (KCa) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine
coronary arteries and smooth muscle cells to acidosis, as well as the role of KCa channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution,
elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly
inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When
we exposed porcine coronary artery smooth muscle cells to a low-pH solution, KCa channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca2+ chelator for which the cell membrane is permeable, abolished the H+-mediated activation of KCa channels in cell-attached patches. Under these circumstances H+ actually inhibited KCa channel activity. When inside-out patches were exposed to a [Ca2+] of 10–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], KCa channels were activated by H+ concentration dependently. However, when these patches were exposed to a [Ca2+] of 10–6 M adjusted with BAPTA at pH 7.3, H+ inhibited KCa channel activity. Extracellular acidosis had no significant direct effect on KCa channels, suggesting that extracellular H+ exerts its effects after transport into the cell, and that KCa channels are regulated by intracellular H+ and by cytosolic free Ca2+ modulated by acute acidosis. These results indicate that the modulation of KCa channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial
tone.
Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998 相似文献
17.
Péter Várnai Gábor L. Petheö Judit K. Makara András Spät 《Pflügers Archiv : European journal of physiology》1998,435(3):429-431
Elevation of extracellular potassium concentration by as little as some tenth of mM activates rat adrenal glomerulosa cells.
In the present study some factors responsible for this high K+ sensitivity were examined. Using whole-cell voltage-clamp technique we found that both T-type and L-type voltage-dependent
Ca2+ channels have very low threshold potential (–71 and –58 mV, resp.). By means of patch-clamp technique combined with single-cell
fluorimetry we also provided evidence that the activation of Igl, a K+-activated inward rectifying current is associated with Ca2+ influx. Both the low activation threshold of voltage-dependent Ca2+ channels and the function of Igl contribute to the exceptional K+ sensitivity of the glomerulosa cells.
Received: 30 September 1997 / Accepted: 4 November 1997 相似文献
18.
Hanns Ulrich Zeilhofer Thomas H. Müller Dieter Swandulla 《Pflügers Archiv : European journal of physiology》1996,432(2):248-257
The contribution of L-, N-, P- and Q-type Ca2+ channels to excitatory and inhibitory synaptic transmission and to whole-cell Ba2+ currents through Ca2+ channels (Ba2+ currents) was investigated in rat hypothalamic neurons grown in dissociated cell culture. Excitatory and inhibitory postsynaptic
currents (EPSCs and IPSCs) were evoked by stimulating individual neurons under whole-cell patch-clamp conditions. The different
types of high-voltage-activated (HVA) Ca2+ channels were identified using nifedipine, ω-Conus geographus toxin VIA (ω-CTx GVIA), ω-Agelenopsis aperta toxin IVA (ω-Aga IVA), and ω-Conus magus toxin VIIC (ω-CTx MVIIC). N-, but not P- or Q-type Ca2+ channels contributed to excitatory as well as inhibitory synaptic transmission together with Ca2+ channels resistant to the aforementioned Ca2+ channel blockers (resistant Ca2+ channels). Reduction of postsynaptic current (PSC) amplitudes by N-type Ca2+ channel blockers was significantly stronger for IPSCs than for EPSCs. In most neurons whole-cell Ba2+ currents were carried by L-type Ca2+ channels and by at least two other Ca2+ channel types, one of which is probably of the Q-type and the others are resistant Ca2+ channels. These results indicate a different contribution of the various Ca2+ channel types to excitatory and inhibitory synaptic transmission and to whole-cell currents in these neurons and suggest
different functional roles for the distinct Ca2+ channel types.
Received: 15 November 1995/Accepted: 30 January 1996 相似文献
19.
In electrically non-excitable cells, Ca2+ entry is mediated predominantly by the store-operated Ca2+ influx pathway, which is activated by emptying the intracellular Ca2+ stores. Just how the Ca2+ content of the stores is communicated to the activity of store-operated Ca2+ channels in the plasma membrane is unclear. It has been suggested that, in some cell types, the link is accomplished by either
a small or a heterotrimeric GTP-binding protein, which is inhibited by guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]) and, in some cases, pertussis toxin. Using the whole-cell patch-clamp technique to directly measure the store-operated
Ca2+ current I
CRAC (Ca2+-release-activated Ca2+ current) in RBL cells, we report that manipulations designed to interfere with GTP-binding protein activity (dialysis with
GTP[γ-S], exposure to pertussis toxin) routinely fail to affect the activation of I
CRAC. However, these agents alter the activity of a K+ current in the same cells, demonstrating biological activity. Furthermore, activation of I
CRAC does not seem to require the presence of a pre-existing diffusible messenger in the cytoplasm to any appreciable extent because
the current reaches the same amplitude irrespective of the whole-cell dialysis time. We conclude that neither a mobile pre-existing
molecule nor a GTP-dependent step is necessary for the activation of I
CRAC in RBL-1 cells.
Received: 9 September 1998 / Received after revision and accepted: 2 November 1998 相似文献
20.
Separation of calcium channel current components in mouse chromaffin cells superfused with low- and high-barium solutions 总被引:3,自引:0,他引:3
Jesús M. Hernández-Guijo Ricardo de Pascual Antonio G. García L. Gandía 《Pflügers Archiv : European journal of physiology》1998,436(1):75-82
This study was carried out to characterize the set of voltage-dependent Ca2+ channel subtypes expressed by mouse adrenal chromaffin cells superfused with solutions containing low (2 mM) or high (10 mM)
Ba2+ concentrations. Using 50-ms test pulses at 0 mV from a holding potential of –80 mV, averaged peak current in 10 mM Ba2+ was around 1 nA, and in 2 mM Ba2+ 0.36 nA. When using 2 mM Ba2+ as the charge carrier, nifedipine (3 μM) blocked I
Ba by 40–45%. ω-Conotoxin GVIA (1 μM) caused 26% inhibition, while ω-conotoxin MVIIC (3 μM) produced a 48% blockade. At low
concentrations (20 nM), ω-agatoxin IVA caused 5–15% of current inhibition, while 2 μM gave rise to a 35–40% blockade. In 10 mM
Ba2+, the blocking effects of nifedipine (40%) and ω-conotoxin GVIA (25%) were similar to those seen in 2 mM Ba2+. In contrast, blockade by ω-conotoxin MVIIC was markedly reduced in 10 mM Ba2+ (20–25%) as compared to 10 mM Ba2+ (48%). The blocking actions of ω-agatoxin IVA (2 μM) were also slowed down in 10 mM Ba2+, though the final blockade was unaffected. In 2 mM Ba2+, I
Ba was quickly inhibited by over 94% with combined nifedipine + ω-conotoxin MVIIC + ω-conotoxin GVIA; in 10 mM Ba2+, I
Ba was blocked by 70% with this combination. The data suggest that mouse chromaffin cells express L-type (40%) as well as non-L-type
(60%) high-threshold voltage-dependent Ca2+ channels. The current carried by non-L-type Ca2+ channels consists of about 25% N-type and 35% P/Q-type; P-type channels, if anything, are poorly expressed. The data also
indicate that the fraction of current blocked by ω-conotoxin MVIIC and ω-agatoxin IVA might considerably change as a function
of the Ba2+ concentration of the extracellular solution; taking this fact into consideration, it seems that a residual R-type current
is not expressed in mouse chromaffin cells.
Received: 21 January 1998 / Received after revision and accepted: 20 February 1998 相似文献