|AGB/ARG Basic & Protocols|
|Guanidines as channel permeant probes
AGB (1-amino-4-guanidobutane, agmatine) and ARG ([1-carboxy,1-amino]-4-guanidobutane, L-arginine) are guanidinium analogues of biological origin. Endogenous levels are generally very low in the CNS and retinas of vertebrates, although arginine levels may be elevated in some astrocytes, non-neural epithelial cells and connective tissue elements. However both ARG and AGB display the ability to permeate ionotropic glutamate gated AMPA, KA and NMDA receptor/channels with differential effectiveness. AGB permeates all three to varying degrees, mGluR6-activated channels in retina, and, in addition, seems to permeate some additional nonselective channels in some receptor cells in retina, olfactory epithelium, gustatory epithelium and lateral line, as well as stretch-activated channels in some glia. ARG permeates NMDA and a subset of AMPA receptor/channel complexes but does not measurably permeate cyclic nucleotide gated channels. Furthermore, ARG permeation seems to lead to substantial cytotoxicity in certain cells, perhaps those containing high levels of NO synthase. AGB seems much less cytotoxic. In some tissues, glia display low affinity ARG transport. There is also evidence of AGB transport by low-affinity organic cation transporter 2 (OCT2) and extraneuronal monoamine transporter (EMT) expressed in HEK293 cells, but this seems to be of such low flux that it does not likely yield immunodetectable levels of accumulated AGB, even sufficient probe if present and if there are high levels of transporter expression in brain.
Both AGB and ARG are inexpensive and available commercially. AGB is sold by Sigma as agmatine SO4, Cat# A7127. AGB is presumed to be a divalent cation at physiological pH with both the guanidine head and amino tail ionized, but of course the charge is not localized and the head should be viewed as a monovalent cation. Commercial AGB·SO4 may be briefly used at up to 10 mM in vitro without serious concern for anion effects, but longer exposures require normal chloride levels to maintain osmolarity.
We have used BaCl2 + AGB·SO4 precipitation to quickly yield high levels of reasonably pure AGB·Cl2.
The solubility product (Ksp) for BaSO4 is 1.1 · 10-10, and it is effectively insoluble in water.
We use the following method:
This solution should be 250 mM AGB+2 with 425 mM Cl– and 12.5 mM SO4-2. At an in vitro concentration of 25 mM AGB, the SO4-2 level should be about 2.5 mM, which is osmotically negligible. If necessary, the Ba purification can be pushed to 1% by adding 9.9 ml. We have not found this necessary and have also found no physiological evidence of Ba contamination with this procedure, given the high SO4 levels used.
General Strategies for Using AGB/ARG
|Marc Lab Protocol Book PDF containing detailed Marclab retinal dissection methods|
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