Caged compounds
The imployment of caged compounds is widely accepted by scientists to monitor the response of the cells to different stimuli.
The molecule of a caged compound consists of the molecule whose activity must be studied and molecule which temporally masks the active site of the agonist. The binding of the two molecules must be resistant to intracellular environment but sensitive to an external stimulus which allows to liberate the agonist in the desired time and space. The 2-nitrophenyl-etyl is commonly used for this purpose as its bound with a molecule can be easily broken by the irradiation with UV light. In
the Figure the uncaging reaction of an agonist is schematically represented.
In our laboratory the following caged compounds are routinely used:
- Caged InsP3, second messenger, agonist of InsP3 receptors
(experiment with caged InsP3);
- Caged cADPr, second messenger, releases Ca2+ from ryanodine receptors.
- Cages NAADP, recently descovered second messenger, a potent Ca2+ release agonist;
- Caged EGTA (caged Ca2+), which releases Ca2+ into the cell after the uncaging reaction.
The main features of the two delivery methods (classical microinjections and caged compound technique)
are summarized in the following table:
| Microinjection | Uncaging |
| Delivery of the injected substance in one point of the cell |
Delivery occurs ivenly in the entire volume of the cell |
| Agonist propagates by diffusion and this takes time |
Does not need to propagate and is liberated instantly in the entire volume of the cell |
| Gradient of [C] between the the point of injection and final calculated [C] is about 100-fold (if 1% of cell volume injected) |
The calculated [C] corresponds to the [C] of caged compound in the cell |
| The injected amount corresponds to the amount in the cell |
Quite difficult to calculate the final effective concentration of the uncaged substance* |
| The resulting pattern of [Ca2+] in the cytoplasm is influenced by CICR |
Allows to study the most sensitive sites of Ca2+ release in the cell |
| Requires a microinjection system** |
Requires a UV filter and a manual or computer-controlled shutter |
| Virtually every substance can be microinjected |
The family of caged compounds is limited*** |
| Difficult but possible to combinate 2 or even 3 different agonists in one needle**** |
Allows to perform multiple uncaging cycles, but the combination of different agonists up to date is impossible |
* Depends mostly on: (i) concentration of the caged substance in the cytoplasm, (ii) power of the UV light delivered to the cell, and (iii) time and pattern of irradiation
** Delivery of the dye or of the caged compound is usually performed using a separate microinjector in case of oocytes. Cells can be loaded using cell permeant variants of the dyes or caged compounds.
*** It is noteworthy to to point out that this family is continuously growing up and Molecular Probes provides a wide range of caged substances. (NAADP is an exeption).
**** Separated by water for oil soluble substances and by oil for water soluble ones.
Conclusions: those described above are the absolutely different methods of delivery of an agent into the cell, and a researcher must perfectly understand which aspect
of the intracellular Ca2+ release does he want to study.
Microinjection is suitable for the basic characterisation of the agonist-induced Ca2+ release, where the spatial pattern of Ca2+ release is not important.
Caged compound technique to study the spatio-temporal aspects of Ca2+ signalling.
A disadvantage of caged reactives is that the commercially available caged compounds are contaminated by non-caged reactive. This might be a very important point when minor subthreshold amounts of non-caged agonist can desensitize receptors and inhibit response
to the normally uncaged agonist (for instance NAADP).
This is the link to the Caged compounds section of Molecular Probes.
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