Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.13.12.5 (aequorin)
 1,451 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The study compares the decay of intracellular luminescence activity (Lmax), the levels of basal [Ca2+]i in resting platelets, and agonist-induced peak [Ca2+]i-signals in platelets loaded with aequorin using the EGTA-, DMSO- and hypoosmotic shock treatment (HOST)-techniques. The highest load of intracellular aequorin with almost unchanged luminescence activity during 4 h was achieved with HOST. Lmax decreased linearly in EGTA- and HOST-platelets, but the decay rate and the levels of basal [Ca2+]i were significantly lower in HOST-platelets. Platelet aggregation and aequorin-indicated [Ca2+]i-rise induced by thrombin and collagen were similar in EGTA- and HOST-platelets. In HOST-platelets, ADP-induced platelet aggregation was always accompanied by aequorin-signals, while at a similar time point, aequorin-signals were absent in 3 of 5 cases in EGTA-platelets. The initial aequorin loading was highest in DMSO-platelets, but Lmax described an exponential decay, which was most pronounced when DMSO-platelets were maintained in Ca(2+)-free buffer (R2 = 0.86). Agonist-induced platelet aggregation was significantly reduced in DMSO-platelets: thrombin-stimulation was accompanied by a significantly lower and delayed [Ca2+]i-rise and no aequorin-signal was obtained in response to ADP in 3 of 5 cases. The study shows that in addition of being a rapid loading-technique, the criteria of high intracellular aequorin load with low luminescence consumption, low basal [Ca2+]i and completely preserved platelet functions are most convincingly met by the HOST-method.
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PMID:On the significance of different aequorin loading techniques on intracellular aequorin discharge, baseline calcium, platelet aggregation and aequorin-indicated Ca(2+)-transients. 144 May 4

We have looked at different parameters which could modify platelet behaviour during and after aequorin loading in the presence of DMSO. There is a decreased platelet reactivity in response to ADP, PAF and A-23,187 which appears to be mainly due to the exposure to EGTA during washing and loading and the 1 ml volume of the test suspension. All the studied agonists (including PMA) which elicit aggregation are able to induce an intracellular Ca2+ change detected with the aequorin probe. By contrast, epinephrine alone induces neither aggregation nor Ca2+ rise, but potentiates the responses to ADP. Different consecutive phases in Ca2+ changes after stimulation with ADP, PMA and A-23,187 can be evidenced. In the presence of external Ca2+, the second component of the Ca2+ change evoked with ADP is dependent on aggregation and the subsequent TXA2 synthesis. When the external medium is Ca2+ depleted, the two Ca2+ peaks induced by ADP disappear whereas a Ca2+ rise persists (endogenous mobilization) with the other agonists, being independent of TXA2 and ADP release. Ca2+ mobilization parallels activation with A-23,187 but not with low concentrations of thrombin.
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PMID:Rapid aequorin loading into platelets in the presence of DMSO--characteristics of the responses (changes in light transmission and in calcium) to various agonists. 148 53

To investigate changes in Ca2+ concentrations in platelet cytoplasm during the activation, aequorin was loaded into platelets with an incubation of dimethyl sulfoxide (DMSO) in platelet suspension. When washed human platelets (about 5 X 10(9) platelets/microliter) were incubated with 10 microM aequorin and 6% DMSO (final concentrations) for 2 min at room temperature, a part of aequorin penetrated through the platelet membrane into the cytoplasm. The leakage of LDH was very slight indicating membrane damage by DMSO treatment being negligible. The platelet membrane and cytoplasmic components preserved their normal features under electron microscope. Platelet aggregation and ATP release were not affected by the incubation. The amount of permeated aequorin was the largest when DMSO was added stepwisely 6 times for 2 min to reach 6% final concentration. Though about 1/5,000 to 1/10,000 of added aequorin penetrated into platelets, the platelets emitted enough luminescent signals by additions of collagen, thrombin and A 23187. The DMSO method is very simple and can save the incubation time (only 2 min at room temperature) to load aequorin into platelets.
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PMID:Simple method of aequorin loading into platelets using dimethyl sulfoxide. 378 65

Dimethyl sulfoxide (DMSO) is a dipolar aprotic solvent widely used in biological assays. Here, we observed that DMSO enhanced the hypo-osmotically induced increases in the concentration of Ca2+ in cytosolic and nucleic compartments in the transgenic cell-lines of tobacco (BY-2) expressing aequorin.
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PMID:Enhanced elevations of hypo-osmotic shock-induced cytosolic and nucleic calcium concentrations in tobacco cells by pretreatment with dimethyl sulfoxide. 3034 96