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1.
The binding of the Ca2+-channel blocker d-cis-[3H]diltiazem to guinea pig skeletal muscle microsomes is temperature-dependent. At 2°C the KD is 39 nM and Bmax is 11 pmol/mg protein. The binding is fully reversible (K−1 = 0.02 min−1). The binding sites discriminate between the diastereoisomers 1- and d-cis-diltiazem, recognize verapamil, gallopamil and tiapamil, and are sensitive to La3+-inhibition. At 30°C the KD is 37 nM and the Bmax is 2.9 pmol/mg protein. D-cis-diltiazem-labelling is regulated by the 1,4-dihydropyridine Ca2+-channel blockers and a novel Ca2+-channel activator in a temperature-dependent manner. At 30°C an enhancement of d-cis-diltiazem binding by the channel blockers is observed. This is attributed to a Bmax increase. EC50-values for enhancement and the maximal enhancement differ for the individual 1,4-dihydropyridines. At 2°C 1,4-dihydropyridines inhibit d-cis-[3H]diltiazem binding. This is attributed to a Bmax decrease. We have directly labelled one of the drug receptor sites within the Ca2+-channel which can allosterically interact with the 1,4-dihydropyridine binding sites. 相似文献
2.
We have characterized the dopamine D2 receptor photoaffinity probe, [3H]azido-N-methylspiperone ([3H]AMS). In the absence of light, [3H]AMS bound reversibly and with high affinity (Kd 70 pM) to sites in canine striatal membranes and was competitively inhibited by dopaminergic agonists and antagonists with an appropriate D2 receptor specificity. Upon photolysis, [3H]AMS covalently incorporated into a peptide of Mr 92,000 as assessed by fluorography following SDS-polyacrylamide gel electrophoresis. Labelling of this peptide was specifically and stereoselectively blocked by D2 antagonists and agonists. Minor specifically labelled peptides of Mr 70,000-55,000 were observed under some conditions and were the result of proteolytic degradation of the peptide at Mr 92,000. 相似文献
3.
(-)-[3H]Desmethoxyverapamil, a novel Ca2+ channel probe. Binding characteristics and target size analysis of its receptor in skeletal muscle 总被引:5,自引:0,他引:5
(-)-[3H]Desmethoxyverapamil (2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan- 7-aza-9-(3-methoxyphenyl)-nonanhydrochloride) was used to label putative Ca2+ channels in guinea pig skeletal muscle. The binding sites for (-)-[3H]desmethoxyverapamil co-purified with t-tubule membrane markers in an established subcellular fractionation procedure. (-)-[3H]Desmethoxyverapamil bound to partially purified t-tubule membranes with a KD of 2.2 +/- 0.1 nM and a Bmax of 18 +/- 4 pmol/mg membrane protein at 25 degrees C. Binding was stereoselectively inhibited by phenylalkylamine Ca2+ antagonists and in a mixed, non-competitive fashion by the benzothiazepine Ca2+ antagonist d-cis-diltiazem and the 1,4-dihydropyridine Ca2+ antagonist (+)-PN 200-110. Target size analysis of the (-)-[3H]desmethoxyverapamil drug receptor site revealed a molecular mass of 107 +/- 2 kDa. In contrast, the target size of the allosterically coupled benzothiazepine drug receptor site, labelled by d-cis-[3H]diltiazem, was 130.5 +/- 4 kDa (p less than 0.01) and of the 1,4-dihydropyridine binding site 179 kDa, when labelled with [3H]nimodipine. It is concluded that (-)-[3H]desmethoxyverapamil is an extremely useful radioligand for the phenylalkylamine-selective receptor site of the t-tubule localized Ca2+ channel which is allosterically linked to two other distinct drug receptor sites. 相似文献
4.
Eun Hui Lee 《Progress in biophysics and molecular biology》2010,103(1):35-43
When observed under a microscope, skeletal muscle exhibits striations due to the highly organized arrangement of muscle proteins that interact with one another to induce muscle contraction. Muscle contraction requires transient increases in intracellular ‘Ca2+’ concentration. In this review, Ca2+ channels contributing to the functional integrity of intracellular Ca2+-release and extracellular Ca2+-entry during skeletal muscle contraction are reviewed in terms of their properties, newly emerging ancillary proteins to them, and their abnormalities related to human skeletal muscle diseases. Finally, the aim of this review is to show the big picture of the correlation among Ca2+ channels that participate in the Ca2+ homeostasis in skeletal muscle. 相似文献
5.
Iwona Ruchala Vanessa Cabra Ernesto Solis Jr. Richard A. Glennon Louis J. De Felice Jose M. Eltit 《Cell calcium》2014
Monoamine transporters have been implicated in dopamine or serotonin release in response to abused drugs such as methamphetamine or ecstasy (MDMA). In addition, monoamine transporters show substrate-induced inward currents that may modulate excitability and Ca2+ mobilization, which could also contribute to neurotransmitter release. How monoamine transporters modulate Ca2+ permeability is currently unknown. We investigate the functional interaction between the human serotonin transporter (hSERT) and voltage-gated Ca2+ channels (CaV). We introduce an excitable expression system consisting of cultured muscle cells genetically engineered to express hSERT. Both 5HT and S(+)MDMA depolarize these cells and activate the excitation-contraction (EC)-coupling mechanism. However, hSERT substrates fail to activate EC-coupling in CaV1.1-null muscle cells, thus implicating Ca2+ channels. CaV1.3 and CaV2.2 channels are natively expressed in neurons. When these channels are co-expressed with hSERT in HEK293T cells, only cells expressing the lower-threshold L-type CaV1.3 channel show Ca2+ transients evoked by 5HT or S(+)MDMA. In addition, the electrical coupling between hSERT and CaV1.3 takes place at physiological 5HT concentrations. The electrical coupling between monoamine neurotransmitter transporters and Ca2+ channels such as CaV1.3 is a novel mechanism by which endogenous substrates (neurotransmitters) or exogenous substrates (like ecstasy) could modulate Ca2+-driven signals in excitable cells. 相似文献
6.
The goal of this investigation was to develop an assay whereby we could measure changes in ATP, ADP, and phosphocreatine (PCr) during stimulation of the sarcoplasmic reticulum (SR) Ca2+ ATPase. After stopping the enzyme reaction, compounds were extracted by perchloric acid and separated by reversed-phase high-performance liquid chromatography (HPLC). Absorbance of ATP and ADP was monitored at 260 nm, and detection of PCr was done at 205 nm. Chromatograms show that peaks associated with each compound are clearly separated and easily detected. The SR Ca2+ ATPase assay was run for various time periods and using varying free [Ca2+]. The changes in ATP and ADP contents were linear with increasing time and varied as expected with increasing free [Ca2+]. The ATPase activities determined using changes in ATP and ADP were nearly identical to those determined using previously established assays. When PCr was added to the assay, we were able to confirm that the Ca2+ ATPase uses ATP that is synthesized locally from PCr via creatine kinase (CK). The results indicate that this is a valid and reliable method for examining SR Ca2+ ATPase activity and for investigating its interaction with CK. 相似文献
7.
Zaldivar D García MC Sánchez JA 《Biochemical and biophysical research communications》2005,338(3):1572-1577
The actions of the ciliary neurotrophic factor (CNTF) were assessed on adult mouse skeletal muscle L-type Ca2+ currents and on Ca2+ release from sarcoplasmic reticulum. Currents were measured with the whole cell patch clamp technique. Ca2+ signals in response to single action potentials were recorded with Fluo3-AM. CNTF (20 ng/ml) reversibly reduced the amplitude of Ca2+ channel currents by 50% within 15 min. In addition, CNTF greatly increased the rate of inactivation during depolarizing pulses and shifted the steady state inactivation curve by -12 mV. The effects of CNTF were mimicked by the PKC activator PMA and prevented by the PKC-inhibitor chelerythrine. In contrast to the effects on the Ca2+ conductance, charge movement and Ca2+ signals remained unaffected by CNTF. These results suggest that CNTF can rapidly decrease muscle Ca2+ channel currents by promoting inactivation, probably through an intracellular PKC-dependent mechanism. 相似文献
8.
The effects of changes in luminal [Ca2+] have been investigated in sheep skeletal sarcoplasmic reticulum (SR) Ca2+-release channels after activation of the channels by different ligands from the cytosolic side of the channel. Native heavy SR membrane vesicles were incorporated into planar phospholipid bilayers under voltage-clamp conditions. Experiments were carried out in symmetrical 250 mm Cs+. Lifetime analysis indicates that channels activated solely by cytosolic Ca2+ exhibit at least two open and five closed states. The open events are very brief and are close to the minimum resolvable duration. When channels are activated solely by cytosolic Ca2+, luminal Ca2+ does not appear to exert any regulatory effect. The P
0 and duration of the open and closed lifetimes are unchanged. However, if channels are activated by ATP alone or by ATP plus cytosolic Ca2+, increases in luminal [Ca2+] produce marked increases in P
0 and in the duration of the open lifetimes. Our results demonstrate that maximum activation of the skeletal SR Ca2+-release channel by ATP cannot be obtained in the absence of millimolar luminal [Ca2+].We are grateful to the British Heart Foundation for financial support. 相似文献
9.
《Cell calcium》2019
Skeletal muscle deficiency in the 3-phosphoinositide (PtdInsP) phosphatase myotubularin (MTM1) causes myotubular myopathy which is associated with severe depression of voltage-activated sarcoplasmic reticulum Ca2+ release through ryanodine receptors. In the present study we aimed at further understanding how Ca2+ release is altered in MTM1-deficient muscle fibers, at rest and during activation. While in wild-type muscle fibers, SR Ca2+ release exhibits fast stereotyped kinetics of activation and decay throughout the voltage range of activation, Ca2+ release in MTM1-deficient muscle fibers exhibits slow and unconventional kinetics at intermediate voltages, suggestive of partial loss of the normal control of ryanodine receptor Ca2+ channel activity. In addition, the diseased muscle fibers at rest exhibit spontaneous elementary Ca2+ release events at a frequency 30 times greater than that of control fibers. Eighty percent of the events have spatiotemporal properties of archetypal Ca2+ sparks while the rest take either the form of lower amplitude, longer duration Ca2+ release events or of a combination thereof. The events occur at preferred locations in the fibers, indicating spatially uneven distribution of the parameters determining spontaneous ryanodine receptor 1 opening. Spatially large Ca2+ release sources were obviously involved in some of these events, suggesting that opening of ryanodine receptors in one cluster can activate opening of ryanodine receptors in a neighboring one. Overall results demonstrate that opening of Ca2+-activated ryanodine receptors is promoted both at rest and during excitation-contraction coupling in MTM1-deficient muscle fibers. Because access to this activation mode is denied to ryanodine receptors in healthy skeletal muscle, this may play an important role in the associated disease situation. 相似文献
10.
Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs+-conducting medium. We found that the pharmacology, Cs+ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10–7 to 10–3 M Ca2+, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca2+, respectively, the cardiac SR channel remained activated in the presence of high [Ca2+]. In correlation to these studies, [3H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca2+] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [3H]ryanodine binding of cardiac SR at millimolar Ca2+ in the absence of adenine nucleotides.When Ca2+-induced Ca2+ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca2+-induced inactivation at high [Ca2+] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca2+ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca2+-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834. 相似文献
11.
Li H Ding X Lopez JR Takeshima H Ma J Allen PD Eltit JM 《The Journal of biological chemistry》2010,285(50):39171-39179
In the absence of store depletion, plasmalemmal Ca(2+) permeability in resting muscle is very low, and its contribution in the maintenance of Ca(2+) homeostasis at rest has not been studied in detail. Junctophilin 1 knock-out myotubes (JP1 KO) have a severe reduction in store-operated Ca(2+) entry, presumably caused by physical alteration of the sarcoplasmic reticulum (SR) and T-tubule junction, leading to disruption of the SR signal sent by Stim1 to activate Orai1. Using JP1 KO myotubes as a model, we assessed the contribution of the Orai1-mediated Ca(2+) entry pathway on overall Ca(2+) homeostasis at rest with no store depletion. JP1 KO myotubes have decreased Ca(2+) entry, [Ca(2+)](rest), and intracellular Ca(2+) content compared with WT myotubes and unlike WT myotubes, are refractory to BTP2, a Ca(2+) entry blocker. JP1 KO myotubes show down-regulation of Orai1 and Stim1 proteins, suggesting that this pathway may be important in the control of resting Ca(2+) homeostasis. WT myotubes stably transduced with Orai1(E190Q) had similar alterations in their resting Ca(2+) homeostasis as JP1 KO myotubes and were also unresponsive to BTP2. JP1 KO cells show decreased expression of TRPC1 and -3 but overexpress TRPC4 and -6; on the other hand, the TRPC expression profile in Orai1(E190Q) myotubes was comparable with WT. These data suggest that an important fraction of resting plasmalemmal Ca(2+) permeability is mediated by the Orai1 pathway, which contributes to the control of [Ca(2+)](rest) and resting Ca(2+) stores and that this pathway is defective in JP1 KO myotubes. 相似文献
12.
Photoaffinity labelling of androgen receptors with 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one 总被引:1,自引:0,他引:1
A O Brinkmann G G Kuiper W de Boer E Mulder H J van der Molen 《Biochemical and biophysical research communications》1985,126(1):163-169
The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in calf uterus and rat prostate. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of 98 kD. In rat prostate cytosol an androgen receptor with a molecular mass of 46 kD could be photoaffinity labelled with R1881. The photoaffinity labelling procedure described here provides a method for studying the hormone binding domain of androgen receptors in partial purified preparations. 相似文献
13.
Muscimol is one of the most potent agonist ligands at the gamma-aminobutyric acidA (GABAA) receptor. Analysis of its chemical structure showed it to be a candidate for photoaffinity labeling. In practice, UV irradiation at 254 nm both changed the UV spectrum of muscimol and induced an irreversible binding of [3H]-muscimol to rat cerebellar synaptosomal membrane. After 10 min of irradiation, using 10 nM [3H]muscimol, the specific portion of this binding was 270 fmol/mg protein. (Nonspecific binding was defined as that arising in the presence of 1 mM GABA.) Specific binding increased asymptotically up to 100 nM [3H]muscimol. Irradiation of the membranes themselves did not significantly alter the KD or Bmax of reversible [3H]muscimol binding. However, irradiation of [3H]muscimol reduced its capacity subsequently to photolabel the membranes by 86 +/- 3%. Dose-dependent inhibition of binding was observed with muscimol, GABA, and bicuculline methiodide; with 10 nM [3H]muscimol maximum inhibition was 70% of total labeling and the order of potencies of these three compounds was characteristic of labeling to the GABAA receptor. Baclofen, l-glutamate, and diazepam exerted no effect at high concentrations. SDS-PAGE of the photolabeled membranes indicated specific incorporation of radioactivity into two molecular-weight species. One failed to enter the separating gel, implying a molecular weight greater than 250,000 daltons (250 kD). The molecular weight of the other was identified by fluorography to be about 52,000 daltons (52 kD). 相似文献
14.
Falqueto EB Massensini AR Moraes-Santos T Gomez MV Romano-Silva MA 《Cellular and molecular neurobiology》2002,22(5-6):819-826
1. Voltage-gated Na+ channels are responsible for initiation and conduction of action potentials. The arrival of an action potential at nerve terminal increases intracellular Na+ and Ca2+ concentrations. Calcium entry into neurons through voltage-dependent calcium channels is associated with a variety of intracellular processes. Scorpion neurotoxins have been used as tools to investigate mechanisms involved in neurotransmitter release. Tityustoxin (TsTX) is an -type toxin that delays Na+-channel inactivation. Toxin- (TiTX-) is a -type toxin that induces Na+-channel activation at resting potentials.2. In the present work, we describe the effects of both toxins on [3H]acetylcholine ([3H]ACh) release from rat cerebrocortical synaptosomes, in the presence or absence of the calcium channels blockers: -conotoxin-GVIA (-CgTx), 1 M; -agatoxin-IVA (-Aga), 30 nM; -conotoxin-MVIIC (-MVIIC), 1 M; or verapamil, 1M.3. TsTX evokes [3H]ACh release in a concentration-dependent manner with a gradual increase up to saturation at concentrations of 500 nM. However, release of ACh evoked by TiTX- was not linear regarding the toxin concentration. The [3H]-ACh release evoked by TsTX or TiTX- was partially inhibited by -CgTx or -Aga, and blocked with -MVIIC. Verapamil (1 M) had no effect. Tetrodotoxin blocked [3H]ACh release evoked by both toxins.4. These results show that different actions on Na+-channels produce different effects on [3H]ACh release with involvement of distinct presynaptic Ca2+-channels, which supports the idea that sodium channels may modulate neurotransmitter release. 相似文献
15.
Brevetoxin-3 (PbTx-3), described to increase the open probability of voltage-dependent sodium channels, caused trains of action potentials and fast oscillatory changes in fluorescence intensity of fluo-3-loaded rat skeletal muscle cells in primary culture, indicating that the toxin increased intracellular Ca(2+) levels. PbTx-3 did not elicit calcium transients in dysgenic myotubes (GLT cell line), lacking the alpha1 subunit of the dihydropyridine receptor (DHPR), but after transfection of the alpha1DHPR cDNA to GLT cells, PbTx-3 induced slow calcium transients that were similar to those of normal cells. Ca(2+) signals evoked by PbTx-3 were inhibited by blocking either IP(3) receptors, with 2-aminoethoxydiphenyl borate, or phospholipase C with U73122. PbTx-3 caused a tetrodotoxin-sensitive increase in intracellular IP(3) mass levels, dependent on extra-cellular Na(+). A similar increase in IP(3) mass was induced by high K(+) depolarization but no action potential trains (nor calcium signals) were elicited by prolonged depolarization under current clamp conditions. The increase in IP(3) mass induced by either PbTx-3 or K(+) was also detected in Ca(2+)-free medium. These results establish that the effect of the toxin on both intracellular Ca(2+) and IP(3) levels occurs via a membrane potential sensor instead of directly by Na(+) flux and supports the notion of a train of action potentials being more efficient as a stimulus than sustained depolarization, suggesting that tetanus is the physiological stimulus for the IP(3)-dependent calcium signal involved in regulation of gene expression. 相似文献
16.
Saturable binding sites for the labelled calcium antagonist (+/-)[3H]nimodipine were found in guinea-pig hind limb skeletal muscle homogenates. Binding sites were enriched in a microsomal pellet by differential centrifugation of the homogenate. [3H]Nimodipine binding (Kd = 1.5 +/- 0.03 nM, Bmax = 2.1 +/- 0.25 pmol/protein, at 37 degrees C) copurified (6-fold) in this fraction with [3H]ouabain binding (6.6-fold) and 125I-alpha-bungarotoxin binding (5-fold). d-cis-Diltiazem (but not 1-cis-diltiazem) stimulated (+/-) [3H]nimodipine binding (ED50 1 microM) by increasing the Bmax. Binding sites discriminated between the optical enantiomers of 1.4-dihydropyridine calcium antagonists and the optically pure enantiomers of D-600. The data confirm, with biochemical techniques, the presence of 1,4-dihydropyridine and (+/-) D-600 inhibitable calcium channels in skeletal muscle, previously found with electrophysiological techniques. 相似文献
17.
《Cell calcium》2017
The L-type Ca2+ channel or dihydropyridine receptor (DHPR) in vertebrate skeletal muscle is responsible for sensing sarcolemmal depolarizations and transducing this signal to the sarcoplasmic Ca2+ release channel RyR1 via conformational coupling to initiate muscle contraction. During this excitation-contraction (EC) coupling process there is a slow Ca2+ current through the mammalian DHPR which is fully missing in euteleost fishes. In contrast to ancestral evolutionary stages where skeletal muscle EC coupling is still depended on Ca2+-induced Ca2+-release (CICR), it is possible that the DHPR Ca2+ conductivity during mammalian (conformational) EC coupling was retained as an evolutionary remnant (vestigiality). Here, we wanted to test the hypothesis that due to the lack of evolutionary pressure in post-CICR species skeletal muscle DHPR Ca2+ conductivity gradually reduced as evolution progressed. Interestingly, we identified that the DHPR of the early ray-finned fish sterlet (Acipenser ruthenus) is phylogenetically positioned above the mammalian rabbit DHPR which retained robust Ca2+ conductivity, but below the euteleost zebrafish DHPR which completely lost Ca2+ conductivity. Remarkably, our results revealed that sterlet DHPR still retained the Ca2+ conductivity but currents are significantly reduced compared to rabbit. This decrease is due to lower DHPR membrane expression similar to zebrafish, as well as due to reduced channel open probability (Po). In both these fish species the lower DHPR expression density is partially compensated by higher efficacy of DHPR-RyR1 coupling. The complete loss of Po in zebrafish and other euteleost species was presumably based on the teleost specific 3rd round of genome duplication (Ts3R). Ts3R headed into the appearance of two skeletal muscle DHPR isoforms which finally, together with the radiation of the euteleost clade, fully lost the Po. 相似文献
18.
《Cell calcium》2017
Lysosomes and lysosome-related organelles are emerging as intracellular Ca2+ stores and play important roles in a variety of membrane trafficking processes, including endocytosis, exocytosis, phagocytosis and autophagy. Impairment of lysosomal Ca2+ homeostasis and membrane trafficking has been implicated in many human diseases such as lysosomal storage diseases (LSDs), neurodegeneration, myopathy and cancer. Lysosomal membrane proteins, in particular ion channels, are crucial for lysosomal Ca2+ signaling. Compared with ion channels in the plasma membrane, lysosomal ion channels and their roles in lysosomal Ca2+ signaling are less understood, largely due to their intracellular localization and the lack of feasible functional assays directly applied to the native environment. Recent advances in biomedical methodology have made it possible to directly investigate ion channels in the lysosomal membrane. In this review, we provide a summary of the newly developed methods for monitoring lysosomal Ca2+ and ion channels, as well as the recent discovery of lysosomal ion channels and their significances in intracellular Ca2+ signaling. These new techniques will expand our research scope and our understanding of the nature of lysosomes and lysosome-related diseases. 相似文献
19.
The voltage-clamp technique was used to study Ca2+ and Cl– transient currents in the plasmalemma of tonoplast-free and intact Chara corallina cells. In tonoplast-free cells [perfused medium with ethylene glycol bis(2-aminoethyl ether)tetraacetic acid] long-term inward and outward currents through Ca channels consisted of two components: with and without time-dependent inactivation. The voltage dependence of the Ca channel activation ratio was found to be sigmoid-shaped, with about –140-mV activation threshold, reaching a plateau at V>50 mV. As the voltage increased, the characteristic activation time decreased from approximately 103 ms in the threshold region to approximately 10 ms in the positive region. The positive pulse-activated channels can then be completely deactivated, which is recorded by the Ca2+ tail currents, at below-threshold negative voltages with millisecond-range time constants. This tail current is used for fast and brief Ca2+ injection into tonoplast-free and intact cells, to activate the chloride channels by Ca2+ . When cells are perfused with EDTA-containing medium in the presence of excess Mg2+, this method of injection allows the free submembrane Ca2+
concentration, [Ca2+]c, to be raised rapidly to several tens of micromoles per liter. Then a chloride component is recorded in the inward tail current, with the amplitude proportional to
. When Ca2+ is thus injected into an intact cell, it induces an inward current in the voltage-clamped plasmalemma, having activation–inactivation kinetics qualitatively resembling that in EDTA-perfused cells, but a considerably higher amplitude and duration (approximately 10 A m–2 and inact~0.5 s at –200 mV). Analysis of our data and theoretical considerations indicate that the [Ca2+]c rise during cell excitation is caused mainly by Ca2+ entry through plasmalemma Ca channels rather than by Ca2+ release from intracellular stores. 相似文献
20.
David R. Luthin †Kevin S. Lee †David Okonkwo ‡Peijun Zhang ‡ Joel Linden 《Journal of neurochemistry》1995,65(5):2072-2079
Abstract: The A2a adenosine receptor agonist 2-[2-(4-amino-3-iodophenyl)ethylamino]adenosine is a potent coronary vasodilator. The corresponding radioiodinated ligand, [125 I]APE, discriminates between high- and low-affinity conformations of A2a adenosine receptors. In this study, [125 I]APE was used for rapid (24-h) autoradiography in rat brain sections. The pattern of [125 I]APE binding is consistent with that expected of an A2a -selective radioligand. It is highest in striatum, nucleus accumbens, and olfactory tubercle, with little binding to cortex and septal nuclei. Specific [125 I]APE binding to these brain regions is abolished by 1 µ M 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS-21680) but is little affected by 100 n M 8-cyclopentyl-1,3-dipropylxanthine. Conversion of [125 I]APE to the corresponding arylazide results in [125 I]AzPE. The rank-order potency of compounds to compete for [125 I]AzPE binding in the dark is CGS-21680 > d -( R )- N 6 -phenylisopropyladenosine > N 6 -cyclopentyladenosine, indicating that it also is an A2a -selective ligand. Specific photoaffinity labeling by [125 I]AzPE of a single polypeptide (42 kDa) corresponding to A2a adenosine receptors is reduced 55 ± 4% by 100 µ M guanosine 5'- O -(3-thiotriphosphate) and 91 ± 1.3% by 100 n M CGS-21680. [125 I]APE and [125 I]AzPE are valuable new tools for characterizing A2a adenosine receptors and their coupling to GTP-binding proteins by autoradiography and photoaffinity labeling. 相似文献