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1.
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
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.
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
4.
Ca2+-channel-dependent and -independent inhibition of exocytosis by extracellular ATP in voltage-clamped rat adrenal chromaffin cells 总被引:1,自引:1,他引:1
Wonil Lim Sang Jeong Kim Hai Dun Yan J. Kim 《Pflügers Archiv : European journal of physiology》1997,435(1):34-42
Membrane currents and capacitance were measured to examine the effects of extracellular ATP on exocytosis in voltage-clamped
rat adrenal chromaffin cells. ATP reversibly inhibited Ca2+ current (I
Ca) and exocytosis. The dependency of exocytosis on I
Ca evoked by 1-s depolarizations was determined. However, inhibition of exocytosis was 2.6 times larger than that estimated
from the reduction of I
Ca, implying the existence of a Ca2+-channel-independent pathway. This inhibition did not rely on a further reduction of the intracellular Ca2+ concentration spike. ATP reduced the rate of exocytosis induced by clamping the intracellular Ca2+ concentration. Pertussis toxin blocked the inhibitory effects of ATP on I
Ca and exocytosis. Although RB-2, a P2Y antagonist, blocked the inhibitory effect of ATP on I
Ca, RB-2 itself produced large increase or decrease in membrane capacitance. Adenosine inhibited I
Ca via a pertussis-toxin-sensitive pathway but did not significantly inhibit exocytosis. Our data show that extracellular ATP
inhibits exocytosis via inhibition of I
Ca by activation of a pertussis-toxin-sensitive G-protein linked to P2Y receptors. Furthermore, our data strongly suggest that ATP activates another pathway, which is also G-protein dependent and
accounts for the majority of the inhibitory effect of ATP on exocytosis.
Received: 20 February 1997 / Received after revision: 10 July 1997 / Accepted: 23 July 1997 相似文献
5.
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
6.
Fox AP Cahill AL Currie KP Grabner C Harkins AB Herring B Hurley JH Xie Z 《Acta physiologica (Oxford, England)》2008,192(2):247-261
Ca2+ is the most ubiquitous second messenger found in all cells. Alterations in [Ca2+]i contribute to a wide variety of cellular responses including neurotransmitter release, muscle contraction, synaptogenesis and gene expression. Voltage-dependent Ca2+ channels, found in all excitable cells (Hille 1992), mediate the entry of Ca2+ into cells following depolarization. Ca2+ channels are composed of a large pore-forming subunit, called the alpha1 subunit, and several accessory subunits. Ten different alpha1 subunit genes have been identified and classified into three families, Ca(v1-3) (Dunlap et al. 1995, Catterall 2000). Each alpha1 gene produces a unique Ca2+ channel. Although chromaffin cells express several different types of Ca2+ channels, this review will focus on the Cav(2.1) and Cav(2.2) channels, also known as P/Q- and N-type respectively (Nowycky et al. 1985, Llinas et al. 1989b, Wheeler et al. 1994). These channels exhibit physiological and pharmacological properties similar to their neuronal counterparts. N-, P/Q and to a lesser extent R-type Ca2+ channels are known to regulate neurotransmitter release (Hirning et al. 1988, Horne & Kemp 1991, Uchitel et al. 1992, Luebke et al. 1993, Takahashi & Momiyama 1993, Turner et al. 1993, Regehr & Mintz 1994, Wheeler et al. 1994, Wu & Saggau 1994, Waterman 1996, Wright & Angus 1996, Reid et al. 1997). N- and P/Q-type Ca2+ channels are abundant in nerve terminals where they colocalize with synaptic vesicles. Similarly, these channels play a role in neurotransmitter release in chromaffin cells (Garcia et al. 2006). N- and P/Q-type channels are subject to many forms of regulation (Ikeda & Dunlap 1999). This review pays particular attention to the regulation of N- and P/Q-type channels by heterotrimeric G-proteins, interaction with SNARE proteins, and channel inactivation in the context of stimulus-secretion coupling in adrenal chromaffin cells. 相似文献
7.
Ira R. Josephson 《Pflügers Archiv : European journal of physiology》1996,433(3):321-329
It has been reported previously that the β subunit increases both the ionic current and the gating charge movement of the
human cardiac L-type Ca2+ channel α1 subunit, and that steady-state measurements reveal the presence of two distinct components of the charge movement [Josephson
IR, Varadi G (1996) Biophys J 70:1285–1293]. The present work identifies and characterizes the kinetic properties of the components
of the human cardiac L-type Ca channel gating currents (I
g), and determines the relationship of these components to the activation of the Ca channel ionic current (I
Ca). Cloned human cardiac L-type α1+α2+β3 subunits were transiently expressed in HEK293 cells and calcium channel gating currents were recorded following the addition
of 5 mM Co2+. The steady-state charge integrals of the gating currents (Q
ON-V
m) were fit by a sum of two Boltzmann components: Q
ON1, which ranged over more negative potentials, and Q
ON2, which ranged over more positive potentials. The kinetic components of the ON and OFF gating currents were identified using
bi-exponential curve fitting. Reconstruction of the two kinetic components of charge (Q
ONfast and Q
ONslow) yielded distributions that were similar in their voltage dependence and relative proportion to those measured directly by
steady-state integration of Q
ON1 and Q
ON2. Changes in the initial conditions were found to affect Q
ON1 and Q
ON2 differently. The time constants of the ON gating current decays were similar to those of the activation of I
Ca. The results suggest that: (1) the activation of the human cardiac L-type Ca channel involves the movements of at least two,
functionally distinct gating structures; (2) a fast charge movement (≈1/4 of the total charge; Q
ON1 or Q
ONfast) precedes a slower charge movement (≈3/4 of the total charge; Q
ON2 or Q
ONslow); and (3) channel opening is associated with the conformational change(s) producing Q
ONslow.
Received: 7 June 1996 / Received after revision: 24 September 1996 / Accepted: 1 October 1996 相似文献
8.
Kirill I. Kiselyov Svetlana B. Semyonova Anton G. Mamin G. N. Mozhayeva 《Pflügers Archiv : European journal of physiology》1999,437(2):305-314
In the present work, we characterized the receptor properties and the conductive features of the inositol (1,4,5)-trisphosphate
(IP3)-activated Ca2+ channels present in excised plasma-membrane patches obtained from mouse macrophages and A431 cells. We found that the receptor
properties of the channels tested were similar to those of the IP3 receptor (IP3R) expressed in the endoplasmic reticulum (ER) membrane. These properties include activation by IP3, inhibition by heparin, time-dependent inactivation by high IP3 concentrations, activation by guanosine 5′o-thiotriphosphate and regulation by arachidonic acid. On the other hand, in terms
of conductive properties, the channel closely resembles Ca2+-release-activated Ca2+ channels (I
crac). These conductive properties include extremely low conductance (≈1 pS), very high selectivity for Ca2+ over K+ (P
Ca/P
K>1000), inactivation by high intracellular Ca2+ concentration and, importantly, strong inward rectification. Notably, the same channel was activated by: (1) agonists in
the cell-attached mode of channel recording, and (2) cytosolic IP3 after patch excision. Although the possibility cannot be completely excluded that a novel type of IP3R is expressed exclusively in the plasma membrane, in their entirety our findings suggest that the plasma membrane of mouse
macrophages and A431 cells contains I
crac-like Ca2+ channels coupled to an IP3-responsive protein which displays properties similar to those of the IP3R expressed in the ER membrane.
Received: 16 June 1998 / Received after revision: / 24 August 1998 / Accepted: 1 September 1998 相似文献
9.
Marina G. Mozhayeva K. I. Kiselyov 《Pflügers Archiv : European journal of physiology》1998,435(6):859-864
We have recently shown that the Ca2+ response in endothelial cells evoked by readdition of Ca2+ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca2+ release from Ca2+ stores sensitive to both inositol 1,4,5-trisphosphate and ryanodine. The present experiments were performed to determine
whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431,
which has a varied resting [Ca2+]i. We found that the amplitude of the Ca2+ response evoked by Ca2+ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca2+ release, but did positively correlate with the initial [Ca2+]i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca2+ responses) revealed that there is an accelerating rise in [Ca2+]i during the biphasic response. Application of ryanodine during the plateau-shaped Ca2+ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca2+ release, but it caused a further [Ca2+]i rise when [Ca2+]i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition
of Ca2+ after agonist-evoked store depletion can evoke Ca2+-induced Ca2+ release. This indicates that Ca2+ entry may be overestimated by this widely used protocol.
Received: 28 July 1997 / Received after revision: 25 November 1997 / Accepted: 26 November 1997 相似文献
10.
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
11.
Antonio R. Artalejo Antonio G. García Erwin Neher 《Pflügers Archiv : European journal of physiology》1993,423(1-2):97-103
Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca2+]i measurements were used to characterize Ca2+-activated K+ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 M) induced a rise of [Ca2+]i concomitantly with an outward current at holding potentials positive to –80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range –80 mV to –40 mV. Increasing the extracellular K+ concentration to 20 mM at the holding potential of –78 mV was associated with inwardly directed currents during the [Ca2+]i elevations induced either by histamine (10 M) or short voltage-clamp depolarizations. The current reversal potential was close to the K+ equilibrium potential, being a function of external K+ concentration. Current fluctuation analysis suggested a unit conductance of 3–5 pS for the channel that underlies this K+ current. The current could be blocked by apamin (1 M). Whole-cell current-clamp recordings snowed that histamine (10 M) application caused a transient hyperpolarization, which evolved in parallel with the [Ca2+]i changes. It is proposed that a small-conductance Ca2+-activated K+ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species. 相似文献
12.
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
13.
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
14.
Marina G. Mozhayeva 《Pflügers Archiv : European journal of physiology》1996,433(1-2):180-187
Sustained Ca2+ elevation (”Ca2+ response”), caused by subsequent readdition of Ca2+ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca2+-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular
Ca2+ concentration ([Ca2+]i) was between about 50 and 110 nM, a massive Ca2+ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca2+]i of over 110 nM displayed small plateau-like Ca2+ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted,
the dependence of the Ca2+ response amplitude on resting [Ca2+]i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca2+ responses, the amplitudes of which were almost constant and in the same range as resting [Ca2+]i. Ni2+, known to partly block Ca2+ entry, caused no change in the half-decay time of [Ca2+]i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni2+] when added at the peak of the phasic component of the Ca2+ response. However, it lowered the sustained phase of the Ca2+ response by 42%. When applied at the start of the readdition of Ca2+, Ni2+ blocked the phasic component of the Ca2+ response, there being a threefold decrease in the initial rate of [Ca2+]i rise. In cells with a resting [Ca2+]i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca2+ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca2+ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP
3-) sensitive store by a submaximal concentration of agonist (in Ca2+-free medium) was followed, on readdition of Ca2+, by Ca2+ entry, which appeared to trigger IP
3-sensitive Ca2+ release (IICR) which, in turn, initiated Ca2+-sensitive Ca2+ release (CICR), thus resulting in a massive elevation of [Ca2+]i.
Received: 3 July 1996 / Received after revision and accepted: 9 September 1996 相似文献
15.
Takahiro Kanno Sechiko Suga J. Wu Masao Kimura M. Wakui 《Pflügers Archiv : European journal of physiology》1998,435(4):578-580
Intracellular cAMP-dependent modulation of L-type Ca2+ channel activation in cultured rat islet β-cells has been investigated using the patch-clamp whole-cell current recording
mode. The L-type voltage-dependent Ca2+ current (I
Ca) showed a fast activation followed by a slow inactivation, and was sensitive to Ca2+ channel blockers, for example nifedipine. Application of a cAMP analogue, dibutyryl cyclic AMP (db-cAMP), increased the magnitude
of the peak I
Ca in a concentration-dependent manner. Values of the half-activation potentials (V
1/2), taken from activation curves for I
Ca, were –16.7 ± 1.8 and –21.9 ± 3.4 mV (P < 0.05) before and after application of db-cAMP, respectively, with no change of the slope factor (k) or the reversal potential. Pretreatment with a specific protein kinase A antagonist, Rp-cAMP, prevented the potentiating
effect of db-cAMP. These results indicate that in rat islet β-cells, phosphorylation of cAMP-dependent kinase potentiates
the voltage-dependent activation of L-type Ca2+ channels.
Received: 9 September 1997 / Received after revision: 19 November 1997 / Accepted: 21 November 1997 相似文献
16.
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
17.
ATP-induced Ca2+ signals in bronchial epithelial cells 总被引:2,自引:0,他引:2
I. Sienaert S. Huyghe J. B. Parys M. Malfait K. Kunzelmann H. De Smedt G. M. Verleden L. Missiaen 《Pflügers Archiv : European journal of physiology》1998,436(1):40-48
Ca2+-dependent Cl– secretion in the respiratory tract occurs physiologically or under pathophysiological conditions when inflammatory mediators
are released. The mechanism of intracellular Ca2+ release was investigated in the immortalized bronchial epithelial cell line 16HBE14o-. Experiments on both intact and permeabilized
cells revealed that only inositol 1,4,5-trisphosphate (InsP
3) receptors and not ryanodine receptors are involved in intracellular Ca2+ release. The expression pattern of the three InsP
3 receptor isoforms was assessed both at the mRNA and at the protein level. The level of expression at the mRNA level was type
3 (92.5%) >> type 2 (5.4%) > type 1 (2.1%) and this rank order was also observed at the protein level. The ATP-induced Ca2+ signals in the intact cell, consisting of abortive Ca2+ spikes or fully developed [Ca2+] rises and intracellular Ca2+ waves, were indicative of positive feedback of Ca2+ on the InsP
3 receptors. Low Ca2+ concentrations stimulated and high Ca2+ concentrations inhibited InsP
3-induced Ca2+ release in permeabilized 16HBE14o- cells. We localized a cytosolic Ca2+-binding site between amino acid residues 2077 and 2101 in the type-2 InsP
3 receptor and between amino acids 2030 and 2050 in the type-3 InsP
3 receptor by expressing the respective parts of these receptors as glutathione S-transferase fusion proteins in bacteria. We conclude that the InsP
3 receptor isoforms expressed in 16HBE14o- cells (mainly type-3 and type-2) are stimulated by Ca2+ and that this phenomenon contributes to the ATP-induced Ca2+ signals in intact 16HBE14o- cells.
Recieved: 11 September 1997 / Received after revision: 2 January 1998 / Accepted: 21 January 1998 相似文献
18.
Missiaen L Sipma H Parys JB De Smet P Callewaert G Hill E McCarthy TV De Smedt H 《Pflügers Archiv : European journal of physiology》1999,437(5):691-694
There is still no agreement on the mechanism of the intracellular action of low concentrations of inositol 1,4,5-trisphosphate
(IP3). Intracellular Ca2+ stores may transiently release some Ca2+ before they become insensitive to IP3. Alternatively, stores with a low IP3 threshold may lose all their Ca2+ and the others none. We now report that the IP3 threshold was not correlated with the extent of Ca2+ release in permeabilized A7r5 smooth-muscle cells. In contrast, the maximum rate of release, which was changed either by
varying the level of IP3 receptor (IP3R) activation, or by changing the concentration of IP3R at a constant level of IP3R activation, was directly related to the extent of Ca2+ release. We conclude that IP3-induced Ca2+ release reflects partial emptying of the stores and not all-or-none Ca2+ release of separate quanta.
Received: 22 October 1998 / Received after revision: 15 December 1998 / Accepted: 11 January 1999 相似文献
19.
María T. Vega Carlos Villalobos Benito Garrido Luis Gandía Oriol Bulbena Javier García-Sancho Antonio G. García Antonio R. Artalejo 《Pflügers Archiv : European journal of physiology》1994,429(2):231-239
Zn2+ increased the rate of spontaneous release of catecholamines from bovine adrenal glands. This effect was Ca2+ independent; in fact, in the absence of extracellular Ca2+, the secretory effects of Zn2+ were enhanced. At low concentrations (3–10 M), Zn2+ enhanced the secretory responses to 10-s pulses of 100 M 1,1-dimethyl-4-phenylpiperazinium (DMPP, a nicotinic receptor agonist) or 100 mM K+. In the presence of DMPP, secretion was increased 47% above controls and in high-K+ solutions, secretion increased 54% above control. These low concentrations of Zn2+ did not facilitate the whole-cell Ca2+ (I
Ca) or Ba2+ (I
Ba) currents in patch-clamped chromaffin cells. Higher Zn2+ concentrations inhibited the currents (IC50 values, 346 M for I
Ca and 91 M for I
Ba) and blocked DMPP- and K+-evoked secretion (IC50 values, 141 and 250 M, respectively). Zn2+ permeated the Ca2+ channels of bovine chromaffin cells, although at a much slower rate than other divalent cations. Peak currents at 10 mM Ba2+, Ca2+, Sr2+ and Zn2+ were 991, 734, 330 and 7.4 pA, respectively. Zn2+ entry was also evidenced using the fluorescent Ca2+ probe fura-2. This was possible because Zn2+ causes an increase in fura-2 fluorescence at the isosbestic wavelength for Ca2+, i.e. 360 nm. There was a slow resting entry of Zn2+ which was accelerated by stimulation with DMPP or high-K+ solution. The entry of Zn2+ was concentration dependent, slightly antagonized by 1 mM Ca2+ and completely blocked by 5 mM Ni2+. The entry of Ca2+ evoked by depolarization with high-K+ solution was antagonized by Zn2+. We conclude that inhibition by Zn2+ of evoked catecholamine secretion is associated with blockade of Ca2+ entry through Ca2+ channels recruited by DMPP or K+. However, the facilitation of secretion observed at low Zn2+ concentrations, or in the absence of Ca2+, may be exerted at an intracellular site on the secretory machinery. This is plausible because Zn2+ permeates the bovine chromaffin cell Ca2+ channels and in this way gains access to the cytosol. In addition, we have established conditions for measuring Zn2+ transients in fura-2-loaded cells with a very high sensitivity, taking advantage of the high-affinity binding of Zn2+ to fura-2 and the modification of its fluorescence spectrum. 相似文献
20.
David W. Barnett Jun Liu Stanley Misler 《Pflügers Archiv : European journal of physiology》1996,432(6):1039-1046
α-Latrotoxin (α-LT), from black widow spider venom, is a potent enhancer of the spontaneous quantal release of neurotransmitter
from a variety of nerve terminals and clonal neurosecretory cells. Using electrochemical amperometry and estimation of membrane
impedance by phase detection, we present evidence that α-LT induces exocytosis of catecholamines from rat adrenal chromaffin
cells beginning as rapidly as 30 s after close application of the toxin. This release is largely dependent on adequate levels
of extracellular Ca2+ ([Ca2+]o). Lowering [Ca2+]o from 2 mM to ≤ 10–20 μM reduces the α-LT-induced rise in membrane capacitance by at least sixfold, on average, and nearly
abolishes α-LT-induced quantal amperometric events, while still permitting insertion of non-selective cation channels. Based
on these experiments, we argue that the rapid onset of α-LT action in promoting massive quantal release from chromaffin cells
is primarily due to an increase in the Ca2+ permeability of the plasma membrane through non-selective cation channels.
Received: 22 April 1996 / Accepted: 10 June 1996 相似文献