共查询到20条相似文献,搜索用时 15 毫秒
1.
Henning Ursula Wallukat Gerd Holtzhauer Martin 《Molecular and cellular biochemistry》1996,160(1):47-52
In primary cultures of neonatal rat heart cells we found a linear correlation between the number of L-type calcium channel-specific dihydropyridine (DHP) binding sites and spontaneous beating frequency (v).Formation of glycoproteins in tissue culture was suppressed by different inhibitors of N-glycosylation. This inhibition alters to a different extent the binding of the DHP ligand (+)-[methyl-3H]PN 200-110 and v. The most severe but reversible effect occurs at 6 g/ml tunicamycin (Bmax 45% and v 6%, resp., of control), a slight increase in Bmax at 0.1–0.5 mM castanospermine and 0.05–2.5 mM deoxymannojirimycin. The other inhibitors gave no significant alterations of Bmax. 相似文献
2.
Numata T Yoshino M 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2005,175(4):257-263
The single Ca2+ channel activity was obtained from cell-attached patch recordings with the use of pipettes filled with 100 mM Ba2+ as the charge carrier in myocytes isolated from the lateral oviduct of cricket Gryllus bimaculatus. The following results were obtained. (1) The channel had a unitary conductance of 18 pS. (2) The open time histogram of the channel could be fitted with a single exponential while the closed time histogram could be fitted with the sum of two exponentials, suggesting that there are at least one open state and two closed states for this channel. (3) The open probability of the channel increased with increasing membrane depolarization. (4) The mean current reconstructed by averaging individual current trace responses inactivated slowly and the current–voltage relationship for the peak mean current showed a bell-shaped relation. (5) The dihydropyridine (DHP) Ca2+ antagonist, nifedipine, reduced the mean current by increasing the proportion of blank sweeps. On the other hand, the DHP Ca2+ agonist, Bay K 8644, increased the mean current by increasing the mean open-times of the channel. These results confirm a presence of DHP-sensitive L-type Ca2+ channel in myocytes isolated from the lateral oviduct of cricket G. bimaculatus. 相似文献
3.
Ichida S Abe J Zhang YA Sugihara K Imoto K Wada T Fujita N Sohma H 《Neurochemical research》2000,25(12):1629-1635
The characteristics of the inhibitory effect of calcium ion (Ca2+)/calmodulin (CaM) on specific [125I]-omega-conotoxin GVIA (125I--CTX) binding and on the labeling of 125I--CTX to crude membranes from chick brain were investigated. The inhibitory effect of Ca2+/CaM depended on the concentrations of free Ca2+ and CaM. The IC50 values for free Ca2+ and CaM were about 2.0 × 10–8 M and 3.0 g protein/ml, respectively. The inhibitory effect of Ca2+/CaM was attenuated by the CaM antagonists W-7, prenylamine and CaM-kinase II fragment (290–309), but not by the calcineurin inhibitor FK506. Ca2+/CaM also inhibited the labeling of a 135-kDa band (which was considered to be part of N-type Ca2+ channel 1 subunits) with 125I--CTX using a cross-linker. These results suggest that Ca2+/CaM affects specific 125I--CTX binding sites, probably N-type Ca2+ channel 1 subunits, in crude membranes from chick whole brain. 相似文献
4.
Summary The Ca2– entry pathways in the basolateral plasma membrane of the isolated, nonperfused proximal straight tubule (PST) of rabbit kidney were investigated using fura-2 fluorescence microscopy. Under isotonic conditions, reduction of bath [Ca2–] from 1 mM to 1 M caused intracellular free calcium concentration ([Ca2+]i) to fall close to zero. Treatment with 10 M verapamil, a calcium channel blocker, had a similar effect. Treatment with verapamil or low Ca2+ also induced fluctuations in cell volume. However, isotonic treatment with 10 M nifedipine, a dihydropyridine (DHP)-type calcium channel blocker, did not affect [Ca2+]i or cell volume, indicating that the endogenous Ca2+ entry pathway is verapamil-sensitive but DHP-insensitive. When cells were exposed to hypotonic solutions in the presence of 1 mM Ca2+, they swelled and underwent normal RVD while [Ca2+]i increased transiently to a peak before decreasing to a late phase plateau level above the baseline level (see McCarty, N.A., O'Neil, R.G. 1991.J. Membrane Biol.
123:149–160). When cells were swollen in the presence of verapamil or low bath [Ca2+], RVD was abolished and [Ca2+]i fell well below the baseline during the late phase response. In contrast, when cells were swollen in the presence of nifedipine, RVD and the late phase rise in [Ca2+]i were abolished, but [Ca2+]i did not fall below the baseline level in the late phase, indicating that nifedipine inhibited the swelling-induced Ca2+ entry but that Ca2+ entry by another pathway was undisturbed. It was concluded that PST cells are characterized by two Ca2+ permeability pathways in the basolateral membrane. Under both isotonic and hypotonic conditions, Ca2+ entry occurs at a slow rate via a verapamil-sensitive, DHP-insensitive baseline Ca2+ entry pathway. Cell swelling activates a separate DHP-sensitive, verapamil-sensitive Ca2+ entry pathway, which is responsible for the supply of Ca ions to the Ca2+-dependent mechanism by which cell volume regulation is achieved. 相似文献
5.
Takenori Miyamoto Diego Restrepo Edward J. Cragoe Jr. John H. Teeter 《The Journal of membrane biology》1992,127(3):173-183
Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP3 or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP3 were blocked by ruthenium red, a blocker of an IP3-gated Ca2+-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP3 in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP3 or cAMP when the buffering capacity for Ca2+ of the pipette solution was increased, when extracellular Ca2+ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca
i
] that opened Ca2+-activated K+ channels. The results suggest that different conductances modulated by either IP3 or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca2+-activated K+ channels contribute to the termination of the IP3 and cAMP responses.Abbreviations ATP
adenosine 5-triphosphate
- BAPTA
(bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid
- cAMP
adenosine cyclic 3,5-monophosphate
- cGMP
guanosine cyclic 3,5-monophosphate
- CTX
charybdotoxin
- DCB
3,4-dichlorobenzamil
- EDTA
ethylenediaminetetraacetic acid
- EGTA
ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- IP3
inositol-1,4,5-triphosphate
- NMDG
N-methyl-d-glucamine
We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825. 相似文献
6.
Lumenal calcium modulates unitary conductance and gating of a plant vacuolar calcium release channel
The patch clamp technique has been used to investigate ion permeation and Ca2+-dependent gating of a voltage-sensitive Ca2+ release channel in the vacuolar membrane of sugar beet tap roots. Reversal potential measurements in bi-ionic conditions revealed a sequence for permeability ratios of Ca2+ Sr2+ Ba2+ > Mg2+ K+ which is inversely related to the size of the unitary conductances K+ Mg2+ Ba2+ > Sr2+ Ca2+, suggesting that ion movement is not independent. In the presence of Ca2+, the unitary K+ current is reduced in a concentration- and voltage-dependent manner by Ca2+ binding at a high affinity site (K
0.5 = 0.29 mm at 0 mV) which is located 9% along the electric field of the membrane from the vacuolar side. Comparison of reversal potentials obtained under strictly bi-ionic conditions with those obtained in the presence of mixtures of the two ions indicates that the channel forms a multi-ion pore. Lumenal Ca2+ also has an effect on voltage-dependent channel gating. Stepwise increases of vacuolar Ca2+ from micromolar to millimolar concentrations resulted in a dramatic increase in channel openings over the physiological voltage range via a shift in threshold for channel activation to less negative membrane potentials. The steepness of the concentration dependence of channel activation by Ca2+ at –41 mV predicts that two Ca2+ ions need to bind to open the gate. The implications of the results for ion permeation and channel gating are discussed.We thank Ian Jennings for writing and implementing some of the software used in this study and Anna J. Bate for technical assistance. The work was supported by grants from the Biotechnology and Biological Sciences Research Council to E.J. (PDF/14) and DS (PG87/529). 相似文献
7.
Bruce Cherksey Robert Durrie Peter E. Braun Victor S. Sapirstein 《Neurochemical research》1994,19(8):1101-1106
This study reports the analysis of K+ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K+ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K+ channel activity was regulated by Ca2+. Little or no change in activity was observed when Ca2+ was added to thecis side of the bilayer. Addition of 10 M total Ca2+ also resulted in little change in K+ channel activity. However, at 80 M total Ca2+ all K+ channel activity was suppressed along with the activation of a 100 pS Cl– channel. The K+ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K+ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G0, Gs, and Gi and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K+ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase
2–3 cyclic nucleotide phosphohydrolase
- EDTA
ethylenediamine N,N,N,N-tetraacetic acid
- G-protein
GTP(guanosine triphosphate) binding protein
- GTPS
guanosine 5-O-(3-thiotriphosphate)
- MAG
myelin associated glycoprotein
- Na+
K+ ATPase, Na+ and K+ stimulated adenosine triphosphatase
- PLP
myelin proteolipid protein
Special issue dedicated to Dr. Majorie B. Lees. 相似文献
8.
Mikala Gabor Klöckners Udo Varadi Maria Eisfeld Jörg Schwartz Arnold Varadi Gyula 《Molecular and cellular biochemistry》1998,185(1-2):95-109
The involvement of cAMP-dependent phosphorylation sites in establishing the basal activity of cardiac L-type Ca2+ channels was studied in HEK 293 cells transiently cotransfected with mutants of the human cardiac 1 and accessory subunits. Systematic individual or combined elimination of high consensus protein kinase A (PKA) sites, by serine to alanine substitutions at the amino and carboxyl termini of the 1 subunit, resulted in Ca2+ channel currents indistinguishable from those of wild type channels. Dihydropyridine (DHP)-binding characteristics were also unaltered. To explore the possible involvement of nonconsensus sites, deletion mutants were used. Carboxyl-terminal truncations of the 1 subunit distal to residue 1597 resulted in increased channel expression and current amplitudes. Modulation of PKA activity in cells transfected with the wild type channel or any of the mutants did not alter Ca2+ channel functions suggesting that cardiac Ca2+ channels expressed in these cells behave, in terms of lack of PKA control, like Ca2+ channels of smooth muscle cells. 相似文献
9.
Summary K+, Cl–, and Ca2+ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K+, K+ channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K+ channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K+ channels showed a significant discrimination against Na+ and Cl–. The Cl– channel opened at positive vacuolar membrane potentials for cytoplasmic Cl– influx had a high conductance of 110pS in symmetrical 100mM Cl–. When K+ and Cl– channels were excluded from opening, no traces were found of Ca2+ channel activity for vacuolar Ca2+ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca2+ channel which allowed influx of cytoplasmic Ca2+ into the vacuole when the Ca2+ concentration on the cytoplasmic side was high. When Ca2+ was substituted by Ba2+, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba2+Sr2+Ca2+Mg2+=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca2+ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd2+, but was insensitive to La3+, Gd3+, Ni2+, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K+, Cl–, and Ca2+ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells. 相似文献
10.
Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase
In order to investigate the effect of transmembrane Ca2+ gradient on Gs mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and Gs and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca2+ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to Gs and can be further enhanced by the decrease of such Ca2+ gradient within certain range (100 fold) following Ca2+ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca2+ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca2+ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP
adenosine triphosphate
- -AR
-adrenergic receptors
- AC
adenylyl cyclase
- DHA
dihydroalprenolol
- DPH
diphenylhexatriene
- [Ca2+]i
Ca2+ concentration inside proteoliposomes
- [Ca2+]o
Ca2+ concentration outside proteoliposomes
- cAMP
cyclic adenosine monophosphate
- DTT
Dithiothreitol
- FS
fluorescein sulfonate
- Gs
Stimulatory GTP-binding protein
- GTP
guanosine triphosphate
- GTPS
guanosine 5-O-(3-thiotriphosphate)
- kDa
kilodalton
- SDS
sodium dodecyl sulfate
- Tris
N-tris(hydroxymethyl)aminomethane 相似文献
11.
Shcheglovitov A. K. Zhelay T. I. Kondratskii A. P. Naidenov V. G. Shuba Ya. M. 《Neurophysiology》2004,36(2):93-101
We analyzed the effects of nifedipine on a family of recombinant low-threshold Ca2+ channels functionally expressed in Xenopus oocytes and formed by three different subunits (1G, 1H, and 1I). The 1G and 1I channels demonstrated a low sensitivity to nifedipine even in high concentrations (IC50 = 98 and 243 M, maximum blocking intensity Amax = 25 and 47%, respectively). At the same time, the above agent effectively blocked channels formed by the 1H-subunit (IC50 = 5 M and Amax = 41%). The nifedipine-caused effects were voltage-dependent, and their changes depended on the initial state of the channel. In the case of 1G-subunits, the blockade was determined mostly by binding of nifedipine with closed channels, whereas in the cases of 1H- and 1I-subunits this resulted from binding of nifedipine with channels in the activated and inactivated states. The obtained data allow us to obtain estimates of the pharmacological properties of the above three subtypes of recombinant channels and, in the future, to compare these characteristics with the properties of low-threshold Ca2+ channels in native cells. 相似文献
12.
《Channels (Austin, Tex.)》2013,7(3):243-248
Three physiological functions have been described for the skeletal muscle 1,4-dihydropyridine receptor (CaV1.1): (1) voltage-sensor for excitation-contraction (EC) coupling, (2) L-type Ca2+ channel, and (3) voltage-sensor for slow depolarization-dependent Ca2+ entry. Members of the RGK (Rad, Rem, Rem2, Gem/Kir) family of monomeric GTP-binding proteins are potent inhibitors of the former two functions of CaV1.1. However, it is not known whether the latter function that has been attributed to CaV1.1 is subject to modulation by RGK proteins. Thus, the purpose of this study was to determine whether Rad, Gem and/or Rem inhibit the slowly developing, persistent Ca2+ entry that is dependent on the voltage-sensing capability of CaV1.1. As a means to investigate this question, Venus fluorescent protein-fused RGK proteins (V-Rad, V-Rem and V-Gem) were overexpressed in “normal” mouse myotubes. We observed that such overexpression of V-Rad, V-Rem or V-Gem in myotubes caused marked changes in morphology of the cells. As shown previously for YFP-Rem, both L-type current and EC coupling were also impaired greatly in myotubes expressing either V-Rad or V-Gem. The reductions in L-type current and EC coupling were paralleled by reductions in depolarization-induced Ca2+ entry. Our observations provide the first evidence of modulation of this enigmatic Ca2+ entry pathway peculiar to skeletal muscle. 相似文献
13.
In previous studies cadmium chloride (CdCl2) nonlethally inhibited Y-1 mouse adrenal tumor cell 20-dihydroxyprogesterone (20DHP) secretion, affecting unstimulated and stimulated steroidogenic pathway sites differently. In addition, dibutyryl cAMP-stimulated 20DHP secretion was unaffected by CdCl2, while the site of the unstimulated effect was indirectly shown to involve steps between endogenous cholesterol utilization and 20-hydroxycholesterol association with mitochondrial cytochrome P450 side-chain cleavage enzyme. In the present study we determined CdCl2 effects on plasma membrane sites preceding pre-dbcAMP-stimulation of 20DHP secretion. Y-1 cells were incubated 0.5 h in medium with or without cadmium (using the concentration that inhibited adrenocorticotropin- (ACTH)-stimulated steroid secretion by 50%) together with exogenously added maximally stimulating concentrations of ACTH, cholera toxin, forskolin, or adenosine triphosphate Cholera toxin, forskolin and ATP bypass specific plasma membrane sites involved in the synthesis of intracellular cAMP and activate the steroid hormone biosynthetic pathway. Cadmium effects on ACTH-stimulated endogenous cAMP secretion were also examined. CdCl2 significantly reduced Y-1 cell 20DHP secretion following exposure to ACTH, cholera toxin, forskolin, and ATP; it also significantly decreased endogenous cAMP secretion into culture medium. These data may be interpreted to suggest that CdCl2 altered Y-1 cell regulation of adenyl cyclase activity, which reduced cAMP-activated cholesterol uptake by mitochondria as a consequence.Abbreviations ACTH
adrenocorticotropin
- ATP
adenosine triphosphate
- ANOVA
analysis of variance
- CdCl2 or Cd2+
cadmium chloride
- cAMP
cyclic 3,5-adenosine monophosphate
- CTX
cholera toxin
- dbcAMP
dibutyryl cAMP,N,O-dibutyryl-3,5-adenosine monophosphate
- EGTA
ethylene glycol bis tetraacetic acid
- FMEM
serum-free Eagle's Minimum Essential Medium with all other supplements
- FSK
forskolin
- Hepes
N-2-hydroxyethylpiperazine-N-1,2-ethanesulfonic acid
- IC50'
concentration inhibiting stimulated steroid secretion by 50%
- IU
international unit
- MEM
Eagle's Minimum Essential Medium
- P450scc
cytochrome P450 side-chain cleavage enzyme
- PREG
pregnenolone
- PROG
progesterone
- SEM
standard error of the mean
- SMEM
serum-containing Eagle's Minimum Essential Medium with supplements
- 20DHP
20--hydroxy-4-pregnen-3-one 相似文献
14.
Taichi Usui Masahiro Suzuki Toshinari Sato Hirokazu Kawagishi Kyoko Adachi Hiroshi Sano 《Glycoconjugate journal》1994,11(2):105-110
Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man
d-mannose
- (M
n) (n=1–5)
-linkedn-mer of mannose
- GlcNAc2
2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose 相似文献
15.
S.A. Mathias O.P. Mgbonyebi E. Motley J.R. Owens J.J. Mrotek 《Cell biology and toxicology》1998,14(3):225-236
In previous studies, nonlethal CdCl2 concentrations apparently inhibited basal Y-1 mouse adrenal tumor cell endogenous mitochondrial cholesterol conversion to pregnenolone. In addition, CdCl2 inhibited all agents stimulating both plasma membrane-dependent cAMP synthesis and 20-hydroxy-4-pregnen-3-one (20DHP) secretion. Bypassing the plasma membrane using dibutyryl-cAMP (dbcAMP) stimulated cytoplasmic cholesterol metabolism and 20DHP secretion in the presence of CdCl2. Since CdCl2 competed at metabolic steps requiring Ca2+ in other tissues, experiments were designed to examine Cd2+ competition with Ca2+ during steroidogenesis. Sets of cells incubated with either medium or adrenocorticotropin (ACTH) with or without CdCl2 were also treated with 0, 1.0, 5.0 or 10.0 mmol/L CaCl2 in the presence or absence of EGTA, a relatively specific Ca2+, but not Cd2+, chelating agent. Another experimental cell set incubated with either medium or ACTH, with or without CdCl2, was treated with or without 1 mmol/L A23187, an ionophore specifically facilitating extracellular Ca2+ transfer across plasma membranes. Besides determining Ca2+ involvement in steroidogenesis using steroid secretion as an endpoint, we directly measured Ca2+ concentrations using intracellular fura-2 fluorescence. Following loading with 2 mol/L fura-2, cells remained untreated or medium was infused with CdCl2, ACTH, ACTH/CdCl2 or ACTH followed after 50 s by CdCl2. Using Ca2+-supplemented media, we observed that Cd2+ inhibition of ACTH-stimulated 20DHP secretion was completely reversed. Standard Ca2+-containing medium supplemented with Ca2+ also enhanced maximally stimulated 20DHP secretion by ACTH. 20DHP secretion by ACTH-treated and ACTH/Cd2+-treated cells was only reduced by EGTA, when Ca2+ was not supplemented. The ionophore A23187 increased basal and ACTH-stimulated 20DHP secretion by Cd2+-treated cells, suggesting that extracellular Ca2+ resources may compete against Cd2+ effects on plasma membrane cAMP synthesis and on basal cholesterol metabolism by mitochondria. No time-dependent change in Ca2+ concentrations occurred within untreated cell suspensions. ACTH stimulation caused a 25 s burst in Ca2+ concentrations before returning to basal, steady-state levels. Cd2+ also stimulated intracellular fura-2 fluorescence. Untreated cell suspensions infused with Cd2+ exhibited a continuous rise in intracellular fluorescence. ACTH/CdCl2-treated cells exhibited a hyperbolic rise in intracellular fluorescence over the 300 s study period. Cells treated with Cd2+ 50 s after ACTH treatment initially exhibited the 25 s fluorescence burst followed by a Cd2+-induced hyperbolic rise in intracellular Cd2+. These fluorescence measurements suggested that cytoplasmic Ca2+ changes do not appear to be necessary for basal 20DHP synthesis and secretion; only a 25 s burst in intracellular Ca2+ is necessary to a slightly higher plateau level for stimulated 20DHP synthesis and secretion. Cd2+ freely enters the cell under basal conditions and Cd2+ entry is accelerated by ACTH stimulation. Data were consistent with Ca2+ being required for optimal stimulated steroid production and Cd2+ probably competing with Ca2+ during basal mitochondrial cholesterol metabolism and plasma membrane ACTH-stimulated cAMP generation. 相似文献
16.
Characterization of a chloride channel reconstituted from cardiac sarcoplasmic reticulum 总被引:1,自引:0,他引:1
We have characterized a voltage-sensitive chloride channel from cardiac sarcoplasmic reticulum (SR) following reconstitution of porcine heart SR into planar lipid bilayers. In 250 mm KCl, the channel had a main conductance level of 130 pS and exhibited two substrates of 61 and 154 pS. The channel was very selective for Cl– over K+ or Na+ (
and
). It was permeable to several anions and displayed the following sequence of anion permeability: SCN– > I– > NO
3
–
Br– > Cl– > f– > HCOO–. Single-channel conductance saturated with increasing Cl– concentrations (K
m= 900 mm and max = 488 pS). Channel activity was voltage dependent, with an open probability ranging from 1.0 around 0 mV to 0.5 at +80 mV. From –20 to +80 mV, channel gating was time-independent. However, at voltages below –40 mV the channel entered a long-lasting closed state. Mean open times varied with voltage, from 340 msec at –20 mV to 6 msec at +80 mV, whereas closed times were unaffected. The channel was not Ca2+-dependent. Channel activity was blocked by disulfonic stilbenes, arylaminobenzoates, zinc, and cadmium. Single-channel conductance was sensitive to trans pH, ranging from 190 pS at pH 5.5 to 60 pS at pH 9.0. These characteristics are different from those previously described for Cl– channels from skeletal or cardiac muscle SR.We thank Dr. Barry Pallotta for help with open and closed intervals analysis and Dr. Gerhard Meissner for his suggestions for the preparation of cardiac sarcoplasmic reticulum membranes. This work was supported by a grant from the National Institutes of Health to R.L.R. and a Student Grant-in-Aid from the American Heart Association, North Carolina affiliate to C.T. R.L.R. is an Established Investigator of the American Heart Association. 相似文献
17.
Sculptoreanu A Abramovici H Abdullah AA Bibikova A Panet-Raymond V Frankel D Schipper HM Pinsky L Trifiro MA 《Molecular and cellular biochemistry》2000,203(1-2):23-31
We have analyzed Ca2+ currents in two neuroblastoma-motor neuron hybrid cell lines that expressed normal or glutamine-expanded human androgen receptors (polyGln-expanded AR) either transiently or stably. The cell lines express a unique, low-threshold, transient type of Ca2+ current that is not affected by L-type Ca2+ channel blocker (PN 200-110), N-type Ca2+ channel blocker (-conotoxin GVIA) or P-type Ca2+ channel blocker (Agatoxin IVA) but is blocked by either Cd2+ or Ni2+. This pharmacological profile most closely resembles that of T-type Ca2+ channels [1-3]. Exposure to androgen had no effect on control cell lines or cells transfected with normal AR but significantly changed the steady-state activation in cells transfected with expanded AR. The observed negative shift in steady-state activation results in a large increase in the T-type Ca2+ channel window current. We suggest that Ca2+ overload due to abnormal voltage-dependence of transient Ca2+ channel activation may contribute to motor neuron toxicity in spinobulbar muscular atrophy (SBMA). This hypothesis is supported by the additional finding that, at concentrations that selectively block T-type Ca2+ channel currents, Ni2+ significantly reduced cell death in cell lines transfected with polyGln-expanded AR. 相似文献
18.
Frans H. M. M. van de Put Joost G. J. Hoenderop Jan Joep H. H. M. De Pont Peter H. G. M. Willems 《The Journal of membrane biology》1993,135(2):153-163
Rabbit pancreatic acinar cells, permeabilized by saponin treatment, rapidly accumulated 3.5 nmol of Ca2+/mg protein in an energy-dependent pool when incubated at an ambient free Ca2+ concentration of 100 nm. Maximal loading of the internal stores was reached at 10 min and remained unchanged thereafter. Complete inhibition of the Ca2+ pump with thapsigargin revealed that this plateau was the result of a steady-state between slow Ca2+ efflux and ATP-driven Ca2+ uptake. Sixty percent of the pool could be released by Ins(1,4,5)P3, whereas GTP released another twenty percent. The striking finding of this study is that the energy-dependent store could also be released by ruthenium red. Uptake experiments in the presence of ruthenium red revealed that the dye, at concentrations below 100 m, selectively reduced the size of the Ins(1,4,5)P3-releasable pool. Ruthenium red had no effect on the half-maximal stimulatory concentration of Ins(1,4,5)P3. At concentrations beyond 100 m, the dye also affected the GTP-releasable pool. Comparison with thapsigargin revealed that ruthenium red released Ca2+ from stores loaded to steady-state at a rate markedly faster than can be explained by inhibition of the ATPase alone. From the data presented, we concluded that ruthenium red selectively releases Ca2+ from the Ins(1,4,5)P3-sensitive store by activating a Ca2+ release channel, whereas Ca2+ release from the GTP-sensitive store is predominantly caused by inhibition of the Ca2+ pump. The postulated ruthenium red-sensitive Ca2+ release channel might be similar to the ryanodine-receptor in muscle.The research of Dr. P.H.G.M. Willems has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences. 相似文献
19.
Noriaki Ikemoto Michel Ronjat Lászró G. Mészáros 《Journal of bioenergetics and biomembranes》1989,21(2):247-266
Recent studies of isolated muscle membrane have enabled induction and monitoring of rapid Ca2+ release from sarcoplasmic reticulum (SR)5
in vitro by a variety of methods. On the other hand, various proteins that may be directly or indirectly involved in the Ca2+ release mechanism have begun to be unveiled. In this mini-review, we attempt to deduce the molecular mechanism by which Ca2+ release is induced, regulated, and performed, by combining the updated information of the Ca2+ release kinetics with the accumulated knowledge about the key molecular components.Abbreviations used: AMP-PCP, adenosine 5-(, -methylenetriphosphate); C1/2, concentration a half-maximal activation or inhibition; Con-A, concanavalin A; DACM,N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide; DCCD, dicyclohexylcarbodiimide; SR, sarcoplasmic reticulum; DHP, dihydropyridine; E-C, excitation-contraction; EP, phosphorylated intermediate of the enzyme; IP3, inositol 1,4,5-trisphosphate; JFM, junctional face membrane;M
r, molecular weight; T-tubule, transverse-tubular system. 相似文献
20.
The effects of eight microelements (I–, BO3
3–, MoO4
2–, Co2+, Cu2+, Mn2+, Fe2+, Zn2+) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I– to 25 M l–1, Cu2+ to 1 M l–1, Co2+ to 2 M l–1 and MoO4
2– to 10 M l–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I– (25 M l–1), Cu2+ (1 M l–1), Co2+ (2 M l–1) and MoO4
2– (10 M l–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l–1, 0.0299%, and 9.15 mg l–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control. 相似文献