ad 1: The localisation of arginine decarboxylase but not agmatine decarboxylase (which to our knowledge does not exist) is one of two central objectives of our study. It is true that the identity and the enzymatic activity of the gene product refered to as arginine decarboxylase (ADC), which was used to generate a polyclonal antibody in our study, is controversially discussed (Coleman et al., 2004; Deignan et al., 2007; Gilad et al., 1996; Li et al., 1995; Lopez-Contreras et al., 2006; Regunathan and Reis, 2000; Zhu et al., 2004). However, there is conclusive evidence justifying the assumption that ADC is a likely candidate to account for arginine decarboxylation, since agmatine was produced in ADC-transfected COS cells (Zhu et al., 2004). Moreover, ornithine decarboxylase (ODC) prefers ornithine as substrate by 1000-fold over arginine (Osterman et al., 1995) and ADC activity was not inhibited by DFMO, a potent and irreversible ODC inhibitor (Li et al., 1995). Also, Kilpelainen and Hietala found that, in the adult rat brain, in contrast to kidney, ODC activity was partially resistant to treatment with DFMO (Kilpelainen and Hietala, 1994). They therefore suggested the existence of an alternative isoform of ODC in the brain. In addition, we performed protein structure and function predictions by submitting the rat ADC sequence (UniProt entry F5A6B1, "Cloning of Rattus norvegicus Arginine Decarboxylase (ADC)." Peters D., Laube G., Veh R.W., Derst C.) to the I-Tasser platform (Zhang, 2008; Roy et al., 2010; Roy et al., 2012). The predicted tertiary structural models are most similar to human ornithine decarboxylase and the highest confidence scores for the Enzyme Classification (EC) number prediction display EC 4.1.1.17 (ornithine decarboxylase) and EC 4.1.1.19 (arginine decarboxylase).

ad 2: A) The role of agmatine as a neurotransmitter/neuromodulator has been reviewed (Halaris and Piletz, 2003) and previously evidence was reported showing that agmatine is contained in rat hippocampal axon terminals associated with synaptic vesicles (Reis et al., 1998) and is released by depolarization of rat brain slices, synaptosome, or bovine adrenal chromaffin cells (Reis and Regunathan, 1998).
B) We did not claim that putrescine derived from agmatine hydrolysis may not be used as a precursor for spermidine/spermine synthesis. Instead, we concluded that, given that many central neurons seem to be able to synthesise polyamines using the classical pathway via ODC, it seems unlikely that the alternative pathway serves this very purpose.

ad 3: see ad 1 and ad 2.

ad 4: Since we did not write a review article on the topic it seems inevitable that not all contributions to the field could be included. However, we apologize to all authors who were not referenced, albeit having made important contributions to polyamine research.

Additional comment: Dr. Gilad has declared no competing interests. However, a conflict of interest may all the same exist, since Dr. Gilad is selling agmatine as a dietary supplement. Thus, a presumed origin of mammalian agmatine from dietary sources (Coleman et al., 2004; Gilad and Gilad, 2013; Piletz et al., 2003) rather than from endogenous synthesis may seem favourable from his point of view.


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