(T1530-05-33) Characterizing the Pharmacological Mechanism of Agmatine in the Spinal Cord Using Calcium Imaging and NMDA-induced Behavioral Assays

Purpose: NMDA receptors (NMDAR) play an essential role in synaptic plasticity, and one of the targets for chronic pain management is antagonism of the NMDARs. Our previous studies showed that agmatine, the decarboxylated form of L-arginine that preferentially antagonizes GluN2B-containing NMDAR, reverses the nerve-injury induced mechanical hypersensitivity without motor side effects (Peterson et al., 2021), and also inhibits NMDAR-mediated excitatory postsynaptic currents in mouse spinal cord dorsal horn (Waataja and Peterson et al., 2019). Agmatine has also been shown to exert its pharmacological effects through multiple sites, including neuronal nitric oxide synthase (nNOS) and organic cation transporters. Nevertheless, the complete pharmacological mechanism of agmatine's attenuation on pain relief remains unknown. The purpose of this study is to elucidate the mechanisms of pain relief by agmatine using calcium imaging and NMDA-induced behavioral assays. We hypothesize that: Agmatine concentration-dependently attenuates NMDAR-mediated Ca²⁺ response. nNOS plays a crucial role in agmatine's inhibition of NDMAR-mediated Ca²⁺ response. PSD95-nNOS inhibitor, IC87201, can reverse the agmatine's analgesic effect on NMDA-induced thermal hyperalgesia.

Methods: Female and male ICR mice (4-6 weeks) were perfused prior to laminectomy and sectioning. The spinal cord slices were loaded with the calcium indicator Fluo-4. Intracellular Ca²⁺ was recorded by single-plane two-photon microscopy. Time-lapse of images were taken and the peak amplitude of fluorescence intensity was analyzed by Student’s t-test and ANOVA. To determine the effect of Agmatine’s inhibition on nNOS, IC87201, a PSD95-nNOS small molecule inhibitor, was co-applied with agmatine in the calcium imaging experiments. For behavior experiments, the NMDA agonist N-methyl-D-aspartate (NMDA) was intrathecally (i.t.) injected in ICR mice (4-7 weeks), which generated thermal hyperalgesia, measured by warm water (49°C) immersion test 5 min after i.t. injection of NMDA. Data are expressed as tail flick latencies (in seconds). The scratching and biting responses in the first minute after NMDA i.t. injection were also measured following the i.t. injection of NMDA.

Results: Bath application of APV (2, 10, 50 µM), ifenprodil (30, 100, 300 µM), and Agmatine (1, 3.3, 10 mM) inhibited the NMDAR-mediated Ca²⁺ response concentration-dependently. The NMDAR-mediated calcium transients in mouse spinal cord dorsal horn were also significantly inhibited following 15 min incubation of 30 µM IC87201, but not with 10 µM IC87201 (Figure 1. A). The increasing concentration of IC87201 (10, 30, 100 µM) concentration-dependently inhibited the NMDAR-mediated calcium response (Figure 1. B). When spinal cord slices were co-incubated in 10 or 30 µM IC87201 with 3.3 mM agmatine, the inhibition of NMDARs-mediated calcium transients by agmatine was significantly reversed with IC87201 (Figure 2.) For NMDA-induced behaviors, 0.01, 0.001, or 0.0001 pmol IC87201 was co-administered with i.t. 1 nmol agmatine and 0.3 nmol NMDA. The inhibition of tail flick latency by i.t. agmatine was significantly reversed with 0.01, 0.001, and 0.0001 pmol IC87201. NMDA-induced nociceptive behaviors, measured in the same animals, were not modulated by 0.01 pmol IC87201 but increased with 0.001 pmol IC87201.

Conclusion: Agmatine inhibited the NMDAR-mediated Ca²⁺ signals concentration-dependently similar to APV and ifenprodil, which supports that agmatine is an effective antagonist of NMDARs in the spinal cord dorsal horn. The PSD95-nNOS inhibitor, IC87201, significantly reversed agmatine’s inhibition on NMDAR-mediated Ca²⁺ response, showing that agmatine’s pharmacological action is mediated both through direct NMDAR antagonism and nNOS inhibition. Agmatine’s analgesic effect on NMDA-induced thermal hyperalgesia is also reversed by IC87201 co-administration, indicating the inhibition of nNOS by agmatine is crucial for agmatine's analgesic effect. These results further define the agmatine’s pharmacological mechanism for pain alleviation, which will help the development of agmatine-based pain treatment.

References: Peterson, Cristina D., Kelley F. Kitto, Harsha Verma, Kelsey Pflepsen, Eric Delpire, George L. Wilcox, and Carolyn A. Fairbanks. 2021. “Agmatine Requires GluN2B-Containing NMDA Receptors to Inhibit the Development of Neuropathic Pain.” Molecular Pain 17 (January): 17448069211029171.
Waataja, Jonathan J., Cristina D. Peterson, Harsha Verma, Cory J. Goracke-Postle, Philippe Séguéla, Eric Delpire, George L. Wilcox, and Carolyn A. Fairbanks. 2019. “Agmatine Preferentially Antagonizes GluN2B-Containing N-Methyl-D-Aspartate Receptors in Spinal Cord.” Journal of Neurophysiology 121 (2): 662–71.

Acknowledgements: This study was supported by DOD Grant W81XWH2010509 to L.V. as well as the College of Pharmacy of the University of Minnesota. No conflicts of interest, financial or otherwise, are declared by the authors.

Figure 1. A) 15 min incubation of 30 µM IC87201 inhibited NMDARs-mediated calcium transients significantly, but not with 10 µM. Symbols represent single cell values, n=2-3 per sex. *p < 0.05 B) 15 min incubation of 10, 30, and 100 µM IC87201 inhibited NMDARs-mediated calcium transients concentration-dependently. Symbols represent mean +/- SEM, n=2-3 per sex.

Figure 2. 10 and 30 µM IC87201 significantly reversed the inhibition of NMDARs-mediated calcium transients by 3.3 mM agmatine. Symbols represent single cell values, and bars represent mean +/- SEM, n=2-3 per sex. *p < 0.05; **p < 0.01