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101.
P. J. Flor J. Gomeza M. A. Tones R. Kuhn J.-P. Pin T. Knöpfel 《Journal of neurochemistry》1996,67(1):58-63
Abstract: The metabotropic glutamate receptor (mGluR) subtype 1 exists as at least three variants (−1a, −1b, and −1c) generated by alternative splicing at the C-terminal domain. Fluorometric Ca2+ measurements were used to compare the concentration dependency of agonist-induced rises in intracellular free Ca2+ concentration ([Ca2+ ]i ) in human embryonic HEK 293 cells transiently expressing rat mGluR1a, mGluR1b, or mGluR1c. The rank order of agonist potencies was quisqualate ≫ (2 S, 1' S, 2' S )-2-(carboxycyclopropyl)glycine (L-CCG-I) > (1 S, 3 R )-1-aminocyclopentane-1,3-dicarboxylic acid [(1 S, 3 R )-ACPD] and did not differ among the splice variants. However, agonists were consistently more potent at mGluR1a than at mGluR1c and mGluR1b. In the same system, we characterized the agonist pharmacology of two chimeric rat mGluR3/1 receptors where the first and/or the second intracellular loop(s) and the C-terminal domain were exchanged with the corresponding mGluR1a or mGluR1c sequences and that were previously shown to mediate elevations in [Ca2+ ]i in response to agonists. The potency of agonists was higher at the chimera having the C-terminus of mGluR1a as compared with those having the mGluR1c C-terminus. Both chimeric mGluR3/1 receptors had the same rank order of agonist potencies: L-CCG-I ≫ (1 S, 3 R )-ACPD ∼ quisqualate. These data support the hypothesis that the C-terminal domain of mGluRs plays a role in determining the potency of agonists for inducing mGluR-mediated functional responses. 相似文献
102.
Keitaro Kiyosawa 《Physiologia plantarum》1996,97(1):180-184
Movements of ions are considered to be governed by the electroneutrality rule. Therefore, a cation moving across the cell membrane into the cell either passively or actively should move together with its counterion, an anion, in equal amounts of charge or in exchange for another cation inside the cell. This means that the net influx of the cation in question should be affected by the permeability of its counterion and/or another cation inside the cell. To examine osmotic and ionic regulation in Chara cells, cell fragments of Chara having a lower osmotic pressure than normal (L-cell fragments) were prepared. The L-cell fragments were individually put into various dilute electrolyte solutions and their osmotic potentials were measured with a turgor balance. Concentrations of K+, Na+, Ca2+, Mg2+, Cl?, NO?3. and SO2?4. in the external electrolyte solutions in which L-cells had been incubated were also analysed by ion chromatography. The results showed that in 0.5 mM KCL + 0.1 mM CaCl2 solution, Chara L-cell fragments absorbed K+ and Cl? to maintain electroneutrality and then regained their osmotic potential very rapidly. When the anion was Cl, the cation absorbed at the highest rate was K+ On the other hand, when the cation was K, the anion absorbed at the highest rate was Cl, Other ions Ca2+, SO2?4 and NO?3 showed much less permeability than K+ and Cl ?for the Chara plasma membrane. The conclusion from these findings was that due to the constraint of electroneutral transport, the uptake rate of a salt into L-cells is limited by the permeability of the least permeable ion. 相似文献
103.
A reassessment of the intervention of calmodulin in the regulation of stomatal movement 总被引:5,自引:0,他引:5
Commelina cammunis L., a monocotyledonous plant whose stomata are highly sensitive to calcium ions, was used to study calmodulin (CaM) involvement in stomatal movements. CaM was detected and quantified in guard cell and mesophyll cell protoplasts by western blot and by 45 Ca2+ -overlays. CaM was found to be 3- to 7-fold more abundant on a per protein basis in guard cell than in mesophyll cell protoplasts. Numerous guard cell proteins that bind CaM in a Ca2+ -dependent manner were detected by gold-labelled CaM overlays. Using bioassays with epidermal strips, different CaM-antagonists were found to induce a net stimulation of stomatal opening in darkness or under illumination (trifluoperazine > compound 48/80 ∼ fluphenazine > W7 > W5). As CaM is frequently involved in the regulation of phosphorylation processes, the effects of different inhibitors of protein kinases on stomatal movements were studied. In red plus blue light, a promotion of the stomatal aperture was observed in the nanomolar range with K252a and KT5926 and in the micromolar range with KT5720 ≫ ML7 ∼ ML9 ≫ H7 > KN62. Only the inhibitors with a high specificity for Ca2+ -CaM dependent protein kinases (K252a, KT5926, ML7, ML9) triggered a stomatal opening in darkness and increased stomatal aperture in red plus blue light. Taken together, these data strongly suggest that a Ca2+ - or a Ca2+ -CaM-dependent protein kinase plays a central role in the calcium transduction pathway leading to the maintaining of stomatal closure. 相似文献
104.
Summary Two dynamic changes are associated with the phytochrome-regulated phototropic response in tip cells of the mossCeratodon purpureus: a tip-located gradient shift of chlortetracy-cline (CTC)-stained calcium and a structural reorganization of apical microfilaments (MFs). We examined the interdependence of these processes. Cells were treated with the antimicrotubule drug oryzalin, the antimicrofilament drug cytochalasin-D, and the calcium channel blocker nifedipine. respectively. The effects on phototropic growth, on the structural alignment of the cytoskeleton (microtubules, MTs; microfilaments) and on the distribution of CTC-stained calcium were studied under each of these conditions. In gravitropically growing tip cells the apical MFs form a cortical collar-like structure, consisting of actin bundles with a parallel axial alignment. These MFs point towards the presumptive growing point, a weakly stained region in the tip of the cell from which bundles are absent. MTs are present in the cortex and in the endoplasm of the tip, predominantly oriented longitudinally. The MTs converge within the central apex. The cells show a steep tip-to-base CTC-calcium gradient with its highest signal in the central apex. Destruction of MTs by 1 M oryzalin induces several translocational effects: (i) the growing zone and phototropic outgrowth shift from the apex to subapical parts of the cell; (ii) the structural integrity of the apical MFs and the tip-to-base alignment of the CTC-calcium gradient are disturbed; and (iii) the red light induced gradient shift and the reorientation of MFs proceed in an expanded area spanning from the tip to subapical parts of the cell. Cytochalasin-D (10 g/ml) destroys the MFs. Under these conditions tip growth stops and the phototropic outgrowth is suppressed. The apical MT-structure and the CTC-calcium gradient are not influenced by the agent. Unilateral red light still induces the light-directed translocation of the gradient. Tip cells memorize a unilateral irradiation applied during growth inhibition with cytochalasin-D. After recovery in darkness the cells start to grow in the former light direction. The restoration of the MFs precedes the outgrowth. The structural alignment of the rebuilt actin bundles indicates the future growth direction. The calcium channel blocker nifedipine (10 M) also inhibits tip growth and concurrently phototropic outgrowth. Nifedipine destroys the CTC-calcium gradient and apical MFs; MTs are not influenced by the channel blocker.Abbreviations CTC
chlortetracycline
- DIC
differential interference contrast
- DMSO
dimethyl sulfoxide
- EGTA
ethyleneglycol-bis-(-aminoethylether) N,N,N-N-tetraacetic acid
- MBS
3-maleimidobenzoic acid N-hydroxysuccimide ester
- MF
microfilament
- MT
microtubule
- MTSB
microtubule stabilizing buffer
- PIPES
piperazine-N,N-bis(2-ethane-sulfonic acid)
- RP
rhodamine labeled phalloidin 相似文献
105.
Summary In the present study we have investigated the presence and distribution of calreticulin in plant protoplasts. Calreticulin was purified from plant homogenates using a selective ammonium sulfate precipitation procedure developed for the purification of mammalian calreticulins and shown to bind calcium in45Ca2+ overlay assays. The protein was localized to plant cell endoplasmic reticulum by the indirect immunofluorescence staining of protoplasts with anti-calreticulin antibodies. No calreticulin was observed within large vacuoles. We conclude that calreticulin is present in the endoplasmic reticulum of plant cells, where, by analogy to the mammalian endoplasmic reticulum, it may play a major role in Ca2+ binding and storage.Abbreviations ER
endoplasmic reticulum
- SR
sarcoplasmic reticulum
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- PBS
phosphate-buffered saline 相似文献
106.
M. E. Marsh 《Protoplasma》1996,190(3-4):181-188
Summary Polyanions are postulated intermediates in biomineralization because they sequester large numbers of calcium ions and occur in high concentrations at mineralizing foci in distantly related organisms. In this study mineral ion and polyanion metabolism was examined inPleurochrysis carterae to determine whether polyanions function as intermediate calcium-carriers during coccolith (mineralized scale) formation. In this organism mineralization occurs intracellularly in coccolith-forming saccules, and mature coccoliths are extruded through the plasma membrane into the coccosphere. The polyanions (acidic polysaccharides known as PS-1 and PS-2) are synthesized in medial Golgi cisternae and transported to the coccolith-forming saccule prior to the onset of mineral deposition; they also cover the mineral surface of mature coccoliths. Pulse-chase experiments with45Ca2+ and14CO3
– show the calcium uptake into the coccolith-forming saccule is much slower than carbonate uptake. The extended intracellular half-life of calcium ions destined for the coccosphere suggests that calcium is initially sequestered in more distal Golgi elements (perhaps in association with the polyanions) and enters the coccolith-forming saccule only after passage through the endomembrane system. This is consistent with previous cytochemical studies showing that the polyanions are complexed with calcium prior to mineral deposition. It has been suggested that polyanions may be degraded at the mineralization front in order to free calcium ions for precipitation with available carbonate or phosphate ions. However, this study demonstrates that the polyanions are not degraded; essentially all PS-1 and PS-2 are eventually secreted with the mineral phase into the coccosphere. The kinetics of mineral ion and polyanion secretion are consistent with a polyanion-mediated calcium transport; however, the manner in which calcium might be sequestered by and freed from the polyanions is still obscure.Abbreviations PS-1/2/3
polysaccharide 1/2/3
- EDTA
ethylenediaminetetraacetic acid
- TCA
trichloroacetic acid 相似文献
107.
Mitogen- and isoproterenol-induced changes of [Ca2+]i in T cells attached to a glass substrate were examined. Murine (C57BL/6) splenic T cells were attached to coverslips or 35-mm dishes (MatTek) precoated with Cell Tak® (3.5 µg/cm2). The cells were then loaded with fluorescent dye (2 µg/ml of fura2-AM or fluo3-AM) and changes in [Ca2+]i in a population of cells (using a spectrofluorometer) or in single cells (using a confocal microscope) were measured during continuous superfusion. Population measurements of [Ca2+]i demonstrated that concanavalin A (Con A, 2 or 5 µg/ml) caused an increase in [Ca2+]i that rose to a peak and then declined to a steady state. The concentration-response relationship (0.05–5 µg/ml) had an EC50 of 0.3 µg/ml. Isoproterenol decreased the Con A-induced elevation of steady state [Ca2+]i. In single cell studies, the increase in [Ca2+]i in response to Con A typically occurred in about 50% of the cells in a microscope field, and the delay before activation varied among cells. Taken together, these data demonstrate that Cell Tak® can be used to attach T cells to glass coverslips and will be useful for the study of signaling mechanisms in T cells. 相似文献
108.
Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mm veratridine by immediate trichocyst exocytosis, provided [Ca2+]
o
(extracellular Ca2+ concentration) is between about 10–4 to 10–3
m as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/ freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec.Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca2+
o
or increasing [Ca2+]
o
to 10–2
m inhibit exocytotic membrane fusion. Veratridine does not release Ca2+ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na+
o
for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca2+]
o
) in the somatic, i.e., nonciliary cell membrane and that a Ca2+ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.We thank Dr. C. Kung (Madison, WI) for providing the pawn mutant, Drs. G. Lehle and R. Waldschütz-Schüppel (Konstanz, Germany) for their help with light scattering experiments, and Ms. E. Dassler and D. Bliestle for continuous help during the extensive photographic documentation. This work has been supported by Deutsche Forschungsgemeinschaft, Schwerpunkt Neue mikroskopische Techniken für Biologie und Medizin (grant P178/11) and SFB156/B4. 相似文献
109.
Cl– conductance in cultured embryonic chick cardiac myocytes was characterized using whole-cell patch clamp techniques. Following elimination of cation currents in Na+and K+-free internal and external solutions, the basal whole-cell current was predominantly a Cl– current. Cl–-sensitive current (I
Cl) was defined as the difference between the whole-cell currents recorded in normal and low [Cl–]
o
when measured in the same cell. The whole-cell current in the absence or presence of 10 m cAMP was time independent, displayed outward rectification with the pipette [Cl–] < 40 mm, and was not saturated with a physiological Cl– gradient. The Cl– current was also activated by 1 m forskolin and inhibited by 0.3 mm anthracene-9-carboxylic acid (9-AC). Forskolin was less effective than cAMP (internal dialysis) in activating the Cl– current. The cAMP- or forskolin-activated and basal Cl– current were reasonably fit by the Goldman-Hodgkin-Katz equation. The calculated P
Cl in the presence of cAMP was increased by fiveto sixfold over the basal level. In the presence of 5 mm EGTA to decrease free [Ca2+]
i
, the whole-cell current could not be stimulated by cAMP, forskolin or IBMX (0.1 mm). These data suggest that cultured chick cardiac myocytes have a low basal Cl– conductance, which, as in some mammalian cardiac ventricular myocytes, can be activated by cAMP. However, this study shows that the activation process requires physiological free [Ca2+]
i
.This study was supported by grants from the National Institutes of Health (HL-17670, HL-27105 and HL-07107) for M.L. and by Institutional funds of the University of Arkansas for Medical Sciences for S.L.We thank Meei-Yueh Liu, Kathleen Mitchell, and Shirley Revels for their technical assistance. 相似文献
110.
Potassium and Taurine Release Are Highly Correlated with Regulatory Volume Decrease in Neonatal Primary Rat Astrocyte Cultures 总被引:2,自引:0,他引:2
Domenico Vitarella †Darryl J. DiRisio †Harold K. Kimelberg Michael Aschner 《Journal of neurochemistry》1994,63(3):1143-1149
Abstract: Neonatal rat primary astrocyte cultures were swollen by exposure to hypotonic buffer. Using an electrical impedance method for determination of cell volume coupled with on-line measurements of efflux of radioactive ions or amino acids, we have investigated the role of K+ (using 86 Rb), taurine, and d -aspartate (an analogue of glutamate) in regulatory volume decrease (RVD). Addition of 1 m M quinine, 10 µ M nimodipine, 100 µ M BAPTA-AM, 10 µ M trifluoperazine, or a calcium-free buffer significantly ( p < 0.0001) inhibited RVD. This was accompanied by inhibition of 86 Rb release but an increase in d -[3 H]-aspartate release, which was proportional to the degree to which RVD was inhibited. These results support a regulatory role for calcium in RVD and show that inhibition of calcium entry from the extracellular fluid, intracellular calcium sequestration, inhibition of calcium-activated K+ channels, and inhibition of calmodulin all inhibit RVD. Because d -[3 H]aspartate efflux profiles increase as RVD is inhibited, it is unlikely that d -aspartate release is a main determinant of RVD. In contrast, [3 H]taurine release was increased by 1 m M quinine and inhibited by 10 µ M trifluoperazine. The net release of K+ and taurine is highly correlated with the degree of RVD, implicating a regulatory role for both K+ and taurine release in RVD. 相似文献