The Marc Laboratory at the University of Utah

University of Utah The JOHN A. MORAN EYE CENTER

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.

Sources
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.

Details

IUPAC name: 1-Amino-4-guanidinobutane Sulfate salt
Molecular Formula H2N(CH2)4NHC(=NH)NH2áH2SO4
Canonical SMILES string C(CCN=C(N)N)CN
Molecular Weight (sulfate) 228.27
Molecular Weight (ion) 130.192
CAS Number 2482-00-0
CID Number CID 199
pKa1 12.5, pKa2 9
AGB pdb file Right-click (Win) or control-click (Mac) & Save As

Preparing AGB·Cl2
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:

Prepare 10 ml of 0.5 M AGB·SO4 (MW 228.27 g/M) in dH₂O
Prepare 10 ml of 0.5 M BaCl2 (MW 244.3 g/M) in dH₂O
Add 9.5 ml of the Ba solution to 10 ml of the AGB in 0.5 ml increments
add & stir each increment 10 sec and allow to settle before adding the next
Pipette off the clear supernatant
Filter through a hard, low ash Whatman filter
Aliquot and store frozen

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

Direct addition of AGB/ARG to salines: In single probe experiments using isolated retinas or brain slices, add 5 mM AGB/ARG for mammalian samples or 10 mM for ectotherm samples to a standard in vitro tissue medium. Proper values are not yet known for all species, but this is a good target level. Incubate tissues for 10 minutes in the presence of desired exogenous agonists/antagonists. Detection of gating of channel permeation by endogenous glutamate takes 20-60 minutes to develop a strong signal.
Modified AGB/ARG salines: Substitute 25 mM NaCl with 25 mM AGB sulfate or ARG chloride. Incubate tissues for about 180 seconds in the presence of desired exogenous agonists/antagonists. Detection of gating of channel permeation by endogenous agents takes 10-20 minutes to develop a strong signal.
Fix with a standard glutaraldehyde/paraformaldehyde fixative (0.1% -2.5% glutaraldehyde + paraformaldehyde). Pre-rinsing is generally not necessary although both AGB and ARG will cross link in the extracellular space around tissue to form a highly immunoreactive matrix. A 60 sec saline rinse will reduce this effect, but it is not necessary.
Process for standard epoxy resin embedding and HPI detection with anti-AGB/anti-ARG IgGs.
anti-AGB IgGs are availible directly from the MarcLab or as cat# ab1568-2000 from Chemicon International (distributor for Signature Immunologics Inc B100 anti-AGB/agmatine IgGs.)
AGB has been tested as an in vivo probe of channel permeation gated by endogenous processes in fishes and mammals. It can be injected in an isotonic medium into the ocular vitreous under anesthesia to achieve 10 mM vitreal levels, followed by a minimum recommended survival time of 45 minutes to achieve good retinal signals. Proper values are not yet known for other species. AGB levels of 3 mM have been used for 10-60 minutes in aquarium water with zebrafish, leading to strong labeling of olfactory receptor cells and mast cells.

Marc Lab Protocol Book PDF containing detailed Marclab retinal dissection methods

Primary References

Marc RE 1999 Mapping glutamatergic drive in the vertebrate retina with a channel permeant organic cation. J Comp Neurol 407:47-64. PubMed PDF

Marc RE 1999 Kainate activation of horizontal, bipolar, amacrine and ganglion cells in the rabbit retina. J Comp Neurol 407:65-76. PubMed PDF

Secondary References

Edwards JG, Michel WC. 2003 Pharmacological characterization of ionotropic glutamate receptors in the zebrafish olfactory bulb. Neuroscience. 122(4):1037-47 PubMed

Edwards JG, Michel WC. 2002 Odor-stimulated glutamatergic neurotransmission in the zebrafish olfactory bulb. J Comp Neurol. 454(3):294-309. PubMed

Kalloniatis, M., Sun, D., Foster, L., Haverkamp, S., & WŠssle, H. 2004. Localization of NMDA receptor subunits and mapping NMDA drive within the mammalian retina. Visual Neurosci., 21, 587-597. PubMed

Lipschitz, D.L. and Michel, W.C. 1999 Physiological evidence for the discrimination
of L-arginine from structural analogs by the zebrafish olfactory system.
J.Neurophysiol. 82: 3160-3167. PubMed

Marc RE and BW Jones 2002 Molecular phenotyping of retinal ganglion cells. J Neurosci 22:413-427. PubMed PDF

Marc RE, BW Jones, CB Watt and E Strettoi 2003 Neural Remodeling in Retinal Degeneration. Progress in Retinal and Eye Research 22: 607-655. PubMed PDF

Michel, W.C. 1999 Cyclic nucleotide-gated channel activation is not required for
activity dependent labeling of zebrafish olfactory receptor neurons by amino
acids. Biological Signals and Receptors 8:338-347 PubMed

Michel WC, Sanderson MJ, Olson JK, Lipschitz DL. 2003 Evidence of a novel transduction pathway mediating detection of polyamines by the zebrafish olfactory system. J Exp Biol. 206(Pt 10):1697-706. PubMed

Michel, W.C., Steullet, P., Cate, H.S., Burns, C.J., Zhainazarov, A.B., and
Derby, C.D. 1999 High-resolution functional labeling of vertebrate and invertebrate
olfactory receptor neurons using agmatine, a channel permeant cation.
J.Neurosci.Meth. 90:143-156 PubMed

Rohrer B, R Blanco, RE Marc, MB Lloyd, D Bok, DM Schneeweis, LF Reichardt 2004 Functionally intact glutamate-mediated signaling in bipolar-cells of the Trkb knockout mouse retina. Visual Neurosci 21:703-13. PubMed PDF

Sakata, Y., Olson, J.K., & Michel, W.C. 2003. Assessment of neuronal maturation and acquisition of functional competence in the developing zebrafish olfactory system. Methods Cell Sci., 25, 39-48. PubMed

Steullet, P., Cate, H.S., Michel, W.C., & Derby, C.D. 2000. Functional units of a compound nose: aesthetasc sensilla house similar populations of olfactory receptor neurons on the crustacean antennule. J Comp Neurol, 418, 270-280. PubMed

Sun D, Kalloniatis M. 2004 Quantification of amino acid neurochemistry secondary to NMDA or betaxolol application. Clin Experiment Ophthalmol. 32(5):505-17. PubMed

Sun, D., Rait, J.L., & Kalloniatis, M. 2003. Inner retinal neurons display differential responses to N-methyl-D-aspartate receptor activation. J Comp Neurol, 465, 38-56. PubMed

Wirsig-Wiechmann CR, Houck LD, Feldhoff PW, Feldhoff RC. 2002 Pheromonal activation of vomeronasal neurons in plethodontid salamanders. Brain Res. 952(2):335-44. PubMed

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