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1.
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
2.
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
3.
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
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.
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
6.
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
7.
Dario A. Protti Osvaldo D. Uchitel 《Pflügers Archiv : European journal of physiology》1997,434(4):406-412
The identity of the voltage-dependent calcium channels (VDCC), which trigger the Ca2+-gated K+ currents (I
K(Ca)) in mammalian motor nerve terminals, was investigated by means of perineurial recordings. The effects of Ca2+ chelators with different binding kinetics on the activation of I
K(Ca) were also examined. The calcium channel blockers of the P/Q family, ω-agatoxin IVA (ω-Aga-IVA) and funnel-web spider toxin
(FTX), have been shown to exert a strong blocking effect on I
K(Ca). In contrast, nitrendipine and ω-conotoxin GVIA (ω-CgTx) did not affect the Ca2+-activated K+ currents. The intracellular action of the fast Ca2+ buffers BAPTA and DM-BAPTA prevented the activation of the I
K(Ca), while the slow Ca2+ buffer EGTA was ineffective at blocking it. These data indicate that P/Q-type VDCC mediate the Ca2+ influx which activates I
K(Ca). The spatial association between Ca2+ and Ca2+-gated K+ channels is discussed, on the basis of the differential effects of the fast and slow Ca2+ chelators.
Received: 20 December 1996 / Received after revision: 21 March 1997 / Accepted: 2 April 1997 相似文献
8.
Opioid potentiation of N-type Ca2+ channel currents via pertussis-toxin-sensitive G proteins in NG108-15 cells 总被引:3,自引:0,他引:3
Hitoshi Morikawa Hiroyuki Mima Hisatoshi Uga Takehiro Shoda Kazuhiko Fukuda 《Pflügers Archiv : European journal of physiology》1999,438(3):423-426
Opioids have both inhibitory and stimulatory effects on neurotransmitter release. While the inhibitory effect has been ascribed
to presynaptic inhibition of Ca2+ channels, the cellular mechanism underlying the stimulatory effect is not clear. In order to address this issue, we analyzed
the effects of [d-Ala2, d-Leu5]-enkephalin (DADLE) on whole-cell Ba2+ currents (I
Ba) through voltage-gated Ca2+ channels in NG108–15 neuroblastoma × glioma hybrid cells. Application of DADLE inhibited and washout of DADLE transiently
potentiated I
Ba. Furthermore, potentiation of I
Ba was elicited even in the presence of DADLE, when inhibition was relieved by a large depolarizing prepulse. DADLE-induced
potentiation, as well as inhibition, had both voltage-sensitive and -insensitive components and was abolished by treatment
with ICI174864, a δ-opioid antagonist, pertussis toxin (PTX) and ω-conotoxin GVIA. Potentiation developed over @3 min and
took 5–20 min to recover, whereas inhibition was complete within 30 s and recovered within 1 min. Although this potentiation
should contribute to DADLE-induced desensitization of Ca2+ channel inhibition, it was not the sole mechanism for desensitization. We conclude that the δ-opioid receptor exerts a dual
action on N-type Ca2+ channels via PTX-sensitive G proteins, i.e., rapid inhibition followed by slowly developing potentiation.
Received: 31 March 1999 / Received after revision: 27 April 1999 / Accepted: 28 April 1999 相似文献
9.
Naoki Kitamura Toshio Ohta Shigeo Ito Y. Nakazato 《Pflügers Archiv : European journal of physiology》1998,435(6):781-788
The mechanisms of depolarizing-prepulse-induced facilitation of Ca2+ channel current were investigated in a study of porcine chromaffin cells. The Ba2+ current evoked by a pulse to 0 mV was increased by a strong depolarizing prepulse (conditioning pulse), termed ”facilitation”.
This facilitation increased with an increase in either the duration or the voltage of the conditioning pulse, and decreased
with an increase in the interpulse interval. For example, the Ba2+ current was increased to 1.14 times the control (facilitation ratio) by a 150-ms conditioning pulse to +100 mV followed by
a 10-ms interpulse interval. Forskolin, 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-bromo-cAMP) and Rp-adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS) did not affect the facilitation of the Ba2+ current, suggesting that a cAMP-dependent mechanism is not involved. Intracellular guanosine 5′-O-(3-thiotriphosphate) (GTPγS) decreased the Ba2+ current to 0.59 times the control and GDPβS increased it to 1.19. However, neither GTPγS nor guanosine 5′-O-(2-thiodiphosphate) (GDPβS) changed the amplitude of the Ba2+ current that was facilitated by the conditioning pulse. Thus, GTPγS increased the facilitation ratio to 2.05 and GDPβS decreased
it to 1.05. Furthermore, the facilitation of the Ba2+ current was abolished by ω-conotoxin GVIA but not by either ω-agatoxin IVA or nifedipine. These results suggest that, in
porcine chromaffin cells, there is a ω-conotoxin GVIA-sensitive N-type Ca2+ channel that is under the inhibitory control of a G protein, which can be relieved by a conditioning pulse.
Received: 25 September 1997 / Received after revision: 14 November 1997 / Accepted: 16 December 1997 相似文献
10.
Just S Heppelmann B 《Experimental brain research. Experimentelle Hirnforschung. Expérimentation cérébrale》2003,150(3):379-384
Voltage-gated Ca2+ channels play an important role in the central processing of nociceptive information. Recently, it has been shown that L-
and N-type voltage-gated Ca2+ channels are also present on peptidergic, fine afferent nerve fibers in the knee joint capsule. Therefore, the influence
of specific blockers for L-type (verapamil) or N-type (ω-conotoxin GVIA) Ca2+ channels on the mechanosensitivity of slowly conducting afferents was tested in the rat knee joint. Topical application of
100 μM verapamil onto the receptive field reduced the mean response to knee joint rotation to 67±8% (SEM, n=12), obtained by outward rotations with a torque of 10 mNm above the mechanical threshold and compared with control movements.
In the presence of 50 μM ω-conotoxin GVIA, the mean response decreased to 44±5% (n=12), a reduction that was also observed during rotations of other intensities. Simultaneous application of both substances
further reduced the response to 25±11% (n=6). In additional experiments it was shown that L- and N-type voltage-gated Ca2+ channels do not influence activity-dependent changes of the mechanical excitability. In conclusion, the data of the present
study indicate that voltage-gated Ca2+ channels may also be involved in the regulation of the mechanosensitivity of nociceptive nerve fiber endings.
Electronic Publication 相似文献
11.
H. Morikawa Kazuhiko Fukuda Hiroyuki Mima Takehiro Shoda Shigehisa Kato Kenjiro Mori 《Pflügers Archiv : European journal of physiology》1998,436(1):127-132
Modulation of Ca2+ channel activity by protein kinases constitutes one of the major mechanisms regulating neuronal functions. Here, we explored
the possible modulation of neuronal Ca2+ channels by protein tyrosine kinases (PTKs). To this end, the effects of PTK inhibitors on whole-cell Ba2+ currents (I
Ba) through voltage-gated Ca2+ channels were analysed in differentiated NG108–15 neuroblastoma × glioma hybrid cells. Genistein suppressed I
Ba in a concentration-dependent fashion (IC50 = 22 μM). Although daidzein, an analogue of genistein that is devoid of PTK inhibitory activity, also suppressed I
Ba, we estimated that specific PTK inhibition by genistein reduced I
Ba amplitude by 30%. In addition, lavendustin A (20 μM) and herbimycin A (20 μM), two other distinct PTK inhibitors, depressed
I
Ba by 22% and 20%, respectively. Genistein suppressed N-type and T-type currents, sparing L-type current, and its effect was
independent of G protein activation. The results suggest that the activity of neuronal Ca2+ channels can be modulated by PTKs, opening the possibility that some of the functions of PTKs in the nervous system are mediated
by Ca2+ channel modulation.
Received: 21 November 1997 / Received after revision: 12 January 1998 / Accepted: 13 January 1998 相似文献
12.
C. Grassi Marcello D’Ascenzo Alessio Valente Gian Battista Azzena 《Pflügers Archiv : European journal of physiology》1999,437(2):241-247
The effect of nitric oxide (NO) donors on high-voltage-activated Ca2+ channels in insulin-secreting RINm5F cells was investigated using the patch-clamp technique in the whole-cell configuration.
Sodium nitroprusside (SNP, 2–400 μM) induced a dose-dependent reduction in Ba2+ currents with maximal inhibition of 58%. The IC50 for SNP was 45 μM. A different NO donor, (±)S-nitroso-N-acetylpenicillamine (SNAP, 500 μM), also produced a 50% decrease in current amplitude. When 200 μM SNP was administered together
with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidozoline-1-oxyl-3-oxide (carboxy-PTIO, 300 μM), the Ba2+ current inhibition was lowered to 7%. Administration of 500 μM 8-bromoguanosine 3′:5′-cyclic monophosphate sodium salt (8-Br-cGMP)
mimicked the effects of SNP, causing a comparable decrease (56%) in peak-current amplitude. When soluble guanylyl cyclase
was blocked by 10 μM 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), the inhibitory effect of 200 μM SNP was reduced from 39% to 15%. The SNP-induced current decrease
was 36% of controls after the blockade of L-type Ca2+ channels and 30% in the presence of 2.5 μM ω-conotoxin-MVIIC. These data indicate that NO inhibits both L-type and P/Q-type
Ca2+ channels in RINm5F cells, probably by an increase in the intracellular levels of cGMP. NO may then significantly influence
the Ca2+-dependent release of hormones from secretory cells as well as that of neurotransmitters from nerve terminals.
Received: 14 April 1998 / Received after revision: 14 September 1998 / Accepted: 25 September 1998 相似文献
13.
Comunanza V Carbone E Marcantoni A Sher E Ursu D 《Pflügers Archiv : European journal of physiology》2011,462(5):709-722
We studied the inhibitory effects of transient receptor potential vanilloid-1 (TRPV1) activation by capsaicin on low-voltage-activated
(LVA, T-type) Ca2+ channel and high-voltage-activated (HVA; L, N, P/Q, R) currents in rat DRG sensory neurons, as a potential mechanism underlying
capsaicin-induced analgesia. T-type and HVA currents were elicited in whole-cell clamped DRG neurons using ramp commands applied
before and after 30-s exposures to 1 μM capsaicin. T-type currents were estimated at the first peak of the I–V characteristics and HVA at the second peak, occurring at more positive potentials. Small and medium-sized DRG neurons responded
to capsaicin producing transient inward currents of variable amplitudes, mainly carried by Ca2+. In those cells responding to capsaicin with a large Ca2+ influx (59% of the total), a marked inhibition of both T-type and HVA Ca2+ currents was observed. The percentage of T-type and HVA channel inhibition was prevented by replacing Ca2+ with Ba2+ during capsaicin application or applying high doses of intracellular BAPTA (20 mM), suggesting that TRPV1-mediated inhibition
of T-type and HVA channels is Ca2+-dependent and likely confined to membrane nano-microdomains. Our data are consistent with the idea that TRPV1-induced analgesia
may derive from indirect inhibition of both T-type and HVA channels which, in turn, would reduce the threshold of nociceptive
signals generation (T-type channel inhibition) and nociceptive synaptic transmission (HVA-channels inhibition). 相似文献
14.
Graça Baltazar Idalina Ladeira Arsélio P. Carvalho Emília P. Duarte 《Pflügers Archiv : European journal of physiology》1997,434(5):592-598
To clarify the role of P-type Ca2+ channels in catecholamine release from adrenal chromaffin cells we examined the concentration dependence of the effect of
ω-agatoxin IVA on the release both of adrenaline and noradrenaline induced by a K+-evoked depolarization. ω-Agatoxin IVA caused a biphasic dose-dependent inhibition of secretion with a high-potency component
(IC50<1 nM), responsible for 10–15% of catecholamine release evoked by 70 mM K+, and a low-potency component that accounted for about 40% of release, with IC50 values of 57 nM and 48 nM for noradrenaline and adrenaline release, respectively. The release of catecholamines from chromaffin
cells was also inhibited dose dependently by ω-conotoxin MVIIC with IC50 values of 182 and 218 nM for noradrenaline and adrenaline release, respectively. The effects of 3 nM ω-agatoxin IVA and 3
μM ω-conotoxin MVIIC were additive, indicating that at the concentrations used the toxins were acting at independent sites,
presumably, P- and Q-type Ca2+ channels. The blockade of Q-type channels inhibited the release of adrenaline (72 ± 4.1%) significantly more than the release
of noradrenaline (50 ± 2.7%), suggesting a higher density or a closer coupling of these channels to exocytosis in adrenergic
chromaffin cells. The blockade of P-type channels caused a greater inhibition of catecholamine secretion at low levels of
K+-evoked depolarization and shorter times of stimulation than that observed at higher levels of stimulation. The contribution
of Q-type channels to catecholamine secretion did not change significantly with the intensity of stimulation. The data show
that two types of ω-agatoxin IVA-sensitive Ca2+ channels are coupled to catecholamine release in chromaffin cells, and that the contribution of P-type channels to secretion
is larger at low levels of depolarization.
Received: 6 March 1997 / Received after revision and accepted: 28 April 1997 相似文献
15.
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
16.
W. Van der Kloot Jordi Molgó Ligia A. Naves 《Pflügers Archiv : European journal of physiology》1997,434(6):735-741
Nicotinic cholinergic agonists are known to decrease synchronous evoked quantal output at the frog neuromuscular junction
[Van der Kloot 1993, J Physiol (Lond) 468:567–589]. Here we also show that carbachol decreases the frequency of miniature
endplate potentials (F
MEPP) in solutions containing elevated levels of K+ and Ca2+. Carbachol did not decrease F
MEPP in hypertonic solutions or in solutions containing the Ca2+ ionophore ionomycin and Ca2+. We conclude that the nicotinic agonists decrease Ca2+ influx through voltage-gated Ca2+ channels. Carbachol did not alter two-pulse facilitation. A blocker of N-type Ca2+ channels, ω-conotoxin GVIA, antagonized the nicotinic agonist-induced decrease in evoked quantal output. The effect of carbachol
was not altered by ω-conotoxin MVIIC, a blocker of P-type and certain other Ca2+channels. The Ca2+ channel targeted by the nicotinic agonists appears to be of the N-type.
Received: 6 March 1997 / Received after revision: 6 May 1997 / Accepted: 4 June 1997 相似文献
17.
Patrizia Tosetti Vanni Taglietti M. Toselli 《Pflügers Archiv : European journal of physiology》1999,437(3):441-448
The ability of action-potential-like waveforms (APWs) to attenuate opioid-induced inhibition of N-type Ca2+ channels was investigated in the neuroblastoma × glioma cell line NG108–15 using whole-cell voltage clamp methods. In in vitro differentiated NG108–15 cells, the opioid agonist [d-ala2]-methionine-enkephalin (DAME) reversibly decreased ω-conotoxin-GVIA-sensitive Ba2+ currents (N-type currents). Agonist-mediated inhibition of N-type currents could be transiently relieved by strong unphysiological
depolarizing prepulses to +80 mV (facilitation). Significant facilitation was also achieved by conditioning the cell with
a train of 15 APWs, which roughly mimicked physiological action potentials (1- to 6-ms-long depolarizations to +30 mV from
a holding potential of –40 mV). The APW-induced facilitation depended on both conditioning pulse frequency and duration. Summation
of the disinhibition produced by each APW was possible because reinhibition following repolarization to –40 mV was a much
slower process (τ=88 ms) than the onset of facilitation at +80 mV (τ=7 ms). These results provide evidence that N-type Ca2+ channel facilitation may be a physiologically relevant process, and suggest that neuronal firing may relieve agonist-induced
inhibition of N-type currents to an extent depending on both the shape of action potentials and the frequency of firing.
Received: 14 September 1998 / Accepted: 29 September 1998 相似文献
18.
Luis Gandía Ricardo Borges Almudena Albillos Antonio G. García 《Pflügers Archiv : European journal of physiology》1995,430(1):55-63
By using the whole-cell configuration of the patch-clamp technique we have investigated the pharmacological properties of Ca2+ channels in short-term cultured rat chromaffin cells. In cells held at a membrane potential of –80 mV, using 10 mM Ba2+ as the charge carrier, only high-voltage-activated (HVA) Ca2+ channels were found. Ba2+ currents (I
Ba) snowed variable sensitivity to dihydropyridine (DHP) Ca2+ channel agonists and antagonists. Furnidipine, a novel DHP antagonist, reversibly blocked the current amplitude by 22% and 48%, at 1 M and 10 M respectively, during short (15–50 ms) depolarizing pulses to 0 mV. The L-type Ca2+ channel agonist Bay K 8644 (1 M) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of I
Ba was accompanied by a 20-mV shift in the activation curves for Ca2+ channels towards more hyperpolarizing potentials. Application of the conus toxin -conotoxin GVIA (GVIA; 1 M) blocked 31% of I
Ba; blockade was irreversible upon removal of the toxin from the extracellular medium, -Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of I
Ba. -Conotoxin MVIIC (MVIIC; 5 M) produced a 36% blockade of I
Ba; such blockade seems to be related to both GVIA-sensitive (N-type) and GVIA-resistant Ca2+ channels. The sequential addition of supramaximal concentrations of furnidipine (10 M), GVIA (1 M), IVA (100 nM) and MVIIC (3 M) produced partial inhibition of I
Ba, which were additive. Our data suggest that the whole cell I
Ba in rat chromaffin cells exhibits at least four components. About 50% of I
Ba is carried by L-type Ca2+ channels, 30% by N-type Ca2+channels and 15% by P-type Ca2+ channels. These figures are close to those found in cat chromaffin cells. However, they differ considerably from those found in bovine chromaffin cells where P-like Ca2+channels account for 45% of the current, N-type carry 35% and L-type Ca2+ channels are responsible for only 20–25% of the current. These drastic differences might have profound physiological implications for the relative contribution of each channel subtype to the regulation of catecholamine release in different animal species. 相似文献
19.
G. J. Stephens Karen M. Page J. Russell Burley Nicholas S. Berrow Annette C. Dolphin 《Pflügers Archiv : European journal of physiology》1997,433(4):523-532
The properties of the rat brain α1E Ca2+ channel subunit and its modulation by accessory rat brain α2-δ and β1b subunits were studied by transient transfection in
a mammalian cell line in order to attempt to reconcile the debate as to whether α1E forms a low-voltage-activated (LVA) or
high-voltage-activated (HVA) Ca2+ channel and to examine its pharmacology in detail. α1E alone was capable of forming an ion-conducting pore in COS-7 cells.
The properties of heteromultimeric α1E/α2-δ/β1b channels were largely dictated by the presence of the β1b subunit, which increased
current density and tended to produce a hyperpolarizing shift in the voltage dependence of activation and inactivation. α1E/α2-δ/β1b
channels did not appear to be regulated by Ca2+-induced inactivation. α1E was shown to exhibit a unique pharmacological profile. ω-Agatoxin IVA blocked the current in a
dose-dependent manner with an IC50 of approximately 50 nM and a maximum inhibition of about 80%, whilst ω-conotoxin MVIIC was without effect. The 1,4-dihydropyridine
(DHP) antagonist nicardipine (1 μM) produced an inhibition of 51 ± 7%, whereas the DHP agonist S-(–)BAY K 8644 was without effect. Our findings suggest a re-evaluation of the classification of the α1E Ca2+ channel subunit; we propose that rat brain α1E forms a novel Ca2+ channel with properties more similar to a subtype of LVA than HVA Ca2+ current.
Received: 30 August 1996 / Received after revision and accepted: 28 October 1996 相似文献
20.
S. L. Soini Emir Duzic Stephen M. Lanier K. E. O. Åkerman 《Pflügers Archiv : European journal of physiology》1997,435(2):280-285
The ability of recombinant rat α2D-and α2B-adrenoceptors expressed in nerve-growth-factor-differentiated pheochromocytoma PC-12 cells to modulate Ca2+ currents, recorded by the whole-cell patch-clamp technique, has been studied. Ca2+ currents in different cells were either reversibly reduced or increased by dexmedetomidine, an α2-adrenergic agonist, in a concentration-dependent manner. Pertussis toxin pretreatment reduced the number of cells that showed
an inhibitory response and reduced the magnitude of inhibition. In cells expressing the α2B-adrenoceptor, pertussis toxin increased the proportion of cells from which a stimulatory effect on Ca2+ currents could be recorded. The magnitude of the inhibitory responses was unaffected but the stimulatory responses were considerably
reduced by the dihydropyridine Ca2+ channel blocker nifedipine (5 μM). All effects of dexmedetomidine were reversible upon wash-out and inhibited by the antagonist
rauwolscine. The results support the idea that modulation of voltage-dependent Ca2+ channels in transfected PC-12 cells is mediated by activation of recombinant α2D- and α2B-adrenoceptors. This receptor activation predominantly causes inhibition of dihydropyridine-insensitive Ca2+ channels via pertussis-toxin-sensitive G proteins. Additionally receptor activation can also lead to stimulation of dihydropyridine-sensitive
Ca2+ channels via pertussis-toxin-insensitive mechanisms.
Received: 25 March 1997 / Received after revision: 27 August 1997 / Accepted: 12 September 1997 相似文献