A study of modulation of P2X3 and TRPV1 receptors by the B-type natriuretic peptide and novel synthetic compounds in trigeminal sensory neurons of wild type and migraine-model mice

Background Trigeminal ganglion (TG) is a key player in processing noxious stimuli. Among many ligand-gated ion channels, trigeminal sensory neurons express on their membranes purinergic P2X3 receptors and capsaicin-sensitive transient receptor potential vanilloid 1 channels (TRPV1). These receptors are thought to be involved in pain transduction and pathophysiology of different pain syndromes, including migraine disorders. P2X3 and TRPV1 channels are continuously regulated by a variety of endogenous modulators, which, upregulating these receptors, can cause sensitization and promote development of pathological pain conditions. Although positive P2X3 and TRPV1 regulators are well studied, not much is known about those which might restrain the activity of these receptors. One candidate for the role of endogenous negative regulator of sensory ganglion activity is the brain natriuretic peptide (BNP). In fact, BNP was recently reported to downregulate inflammatory pain and firing frequency of small neurons in dorsal root ganglia via its receptor NPR-A. Aims In order to investigate the role of BNP/NPR-A system in trigeminal ganglion in control conditions and in migraine pathology we used wild-type (WT) mice and transgenic R192Q KI mice of the familial hemiplegic migraine type 1 (FHM1) model. First we characterized BNP and NPR-A expression and functional properties of the BNP/NPR-A pathway in trigeminal ganglions of WT and KI mice. To understand if this pathway can affect the properties of sensory neurons in TG we studied the effects of endogenous and exogenous BNP on P2X3 and TRPV1 receptors responses in vitro. Investigating molecular mechanisms underneath P2X3 receptor modulation we carefully examined changes in P2X3 phosphorylation and membrane distribution and considered involvement of particular kinases and phosphatases in this process. Firing activity of the WT and KI trigeminal neurons were also evaluated to find out if the modulatory effects of BNP/NPR-A system on the P2X3 channels are reflected in neuronal excitability. Additionally, in search for new potent P2X3 antagonists a variety of diaminopurine derivatives as well as several adenosine nucleotide analogues were evaluated on recombinant P2X3 receptors in HEK cells and on native P2X3 receptors of TG sensory neurons. Results We found abundant expression of NPR-A in trigeminal ganglion along with low levels of BNP itself; the BNP/NPR-A pathway in both WT and KI neurons proved to be functional. Exogenously applied BNP inhibited TRPV1-mediated responses in WT and KI trigeminal neurons without any changes in the receptor’s expression level. On the other hand, P2X3 receptors were not sensitive to additional exogenous BNP, but appeared to be downregulated by the low amount of endogenous BNP already present in WT TG cultures. This negative modulation included P2X3 serine phosphorylation and receptor redistribution to the non-lipid raft membrane compartments. Both mechanisms were dependent on the activity of protein kinase G. Interestingly, in KI mice NPR-A-mediated P2X3 inhibition could not be seen and receptors remained upregulated, most probably due to the increased activity of P/Q calcium channels and high concentration of calcitonin gene related peptide (CGRP). Considering firing properties of trigeminal neurons, inactivation of BNP/NPR-A system with NPR-A antagonist anantin caused a hyperexcitability phenotype of WT cultures, which was very similar to what is typical for KI neurons. KI cultures remained unaltered, consistent with lack of BNP/NPR-A regulation over P2X3 activity. Experiments with new diaminopurine compounds and adenosine nucleotide derivatives resulted in molecules which showed antagonistic behavior towards P2X3 receptors with IC50 values in low micromolar and nanomolar range, respectively. Conclusion The main result of the present study is the identification of BNP/NPR-A pathway as an intrinsic negative modulatory system for P2X3 and TRPV1 receptors activity in sensory neurons of mouse trigeminal ganglion and related neuronal excitability. However, in a mouse FHM1 migraine model BNP/NPR-A lacked the inhibitory effect on P2X3 receptors due to the overall amount of activation these receptors undergo in KI neurons. Modifications of diaminopurine and adenosine scaffold could serve as a promising strategy in search for new potent antagonists of P2X3 receptors.