The first part of the thesis (study I) was carried out in peripheral sensory neurons and host HEK cells to address a potential anti-nociceptive effects of the synthetic di-adenosine tetraphosphates (Ap4A) on pro-nociceptive P2X3 receptors.
5.1.1 Activation and inhibitory effects of Ap4A-analogues on rat recombinant P2X3 receptors
The existing literature suggested that endogenous di-adenosine polyphosphates Ap5A and Ap4A, prone to the hydrolysis with NPTDases, possess the agonist activity on pro-nociceptive P2X3 receptors (Wildman et al., 1999; McDonald et al., 2002). However, the biological activity of the stable resistant to hydrolysis variants of Ap4A was not tested.
Therefore, we explored the agonist and antagonist activity of the stable synthetic Ap4A such as AppNHppA and AppCH2ppA on rat P2X3 (rP2X3) receptors expressed in HEK cells. To this end, we used the whole-cell patch-clamp recordings of membrane currents activated by local fast application of ligands to HEK cells expressing rP2X3 receptors. The reference compound for these experiments was the full agonist of P2X3 receptors -meATP (Sokolova et al., 2006). Our results demonstrated that both AppNHppA and AppCH2ppA were able to activate rP2X3 receptors. However, the agonist activity of these compounds was rather weak as, unlike -meATP, concentrations of either 0.1 or 1 µM of AppNHppA and AppCH2ppA were not sufficient to activate receptor currents (study I, Fig 1 A). Nevertheless, at concentrations of 10 or 100 M AppNHppA was able to activate small and slowly desensitizing currents through rP2X3 receptors (study I, Fig 1 A). To evaluate the agonist activity of the Ap4A-analogues, we compared P2X3 currents induced by AppNHppA and AppCH2ppA with those evoked by the full P2X3 receptors agonist -meATP. We found that both AppNHppA and AppCH2ppA were only partial agonists at rP2X3 receptors (study I, Fig 1 C). Interestingly, the agonist effect of AppCH2ppA was apparently stronger than that of AppNHppA (EC50=9.3±1.7 µM, n=5, versus EC50=41.6±1.3 µM, n=8 respectively, study I, Fig 1 A-C).
One characteristic property of the pro-nociceptive P2X3 receptors is their ability to undergo a long lasting desensitization even without macroscopic activation; a phenomenon called
‘high affinity desensitization’ (HAD) (Sokolova et al., 2006). This mechanism suggests an approach to provide anti-nociception in various pain conditions involving purinergic receptors (Giniatullin and Nistri, 2013).
In order to evaluate the possible anti-nociceptive action of AppNHppA and AppCH2ppA via inhibitory HAD effects on P2X3 receptors, we used following experimental protocol: we applied the P2X3 agonist 10 µMmeATP for 2 s followed by 2 min wash-out before and after AppNHppA or AppCH2ppA administration (0.001-1000 µM concentrations) (study I, Fig 2 ). Notably, AppNHppA applied at a concentration below the activation threshold (100
nM) was able strongly inhibit -meATP-induced currents to 38.2±8.5% (n=7, p=0.002; study I, Fig 2 C). Thus, we demonstrated that AppNHppA possessed an inhibitory effect, with an IC50=0.20±0.04 µM (n=3–6; study I, Fig 2). A similar HAD-mediated inhibitory effect was also observed with AppCH2ppA (IC50=0.55±0.2 µM, n=4–7; study I, Fig 2 A, C-D). More frequent
-meATP application (with 30 s intervals) enhanced the inhibition induced by 1 µM AppCH2ppA from 61% to 81±3% (n=7, p=0.014; study I, Fig 2 B). Thus, the inhibition of the pro-nociceptive P2X3 receptors by AppNHppA or AppCH2ppA was clearly use-dependent.
5.1.2 Selectivity of the AppNHppA and AppCH2ppA effects on the P2X3 receptors
One of key issues in development of the anti-nociceptive agents is the selectivity of their action. By applying the same electrophysiological approach, we further investigated the specificity of Ap4A-analogues for P2X3 receptors. We tested AppNHppA and AppCH2ppA activity on recombinant rP2X2, rP2X4 and rP2X7 receptors. However, we found neither activation nor inhibition by AppNHppA or AppCH2ppA on any of the other tested P2X receptors (study I, Fig 4). Next, to test possible involvement of metabotropic P2Y receptors in P2X3 inhibition, we dialysed cells with GDP-β-S (non-hydrolyzable GDP-analogue, competitive inhibitor of G-proteins, 500 µM) before testing them with AppNHppA and AppCH2ppA. However, GDP-β-S treatment did not change the inhibitory effect of AppNHppA and AppCH2ppA on P2X3 receptor mediated currents (study I, Fig 5). This result indicated that G-protein-coupled P2Y receptors were not involved in the observed P2X3 receptor inhibition. Taken together, our data suggested a selective use-dependent action of Ap4A-analogues on P2X3 receptors mediated via inhibitory HAD mechanism.
5.1.3 Action of the Ap4A-analogues on native P2X3 receptors in sensory neurons
Next, we tested the effects of AppNHppA and AppCH2ppA on native P2X3 receptors expressed in sensory neurons. In this experiment, we used trigeminal ganglion (TG), dorsal root ganglion (DRG) and nodose ganglion (NDG) cultures. These ganglia naturally express either homomeric P2X3 (fast desensitizing component of the current), homomeric P2X2 (slow current component) or variable mixtures of P2X2 and P2X3 subunits (heteromeric P2X2/3 combination generating ‘mixed’ currents with a sustained non-desensitizing component). By applying 2 s applications of α,β-meATP (10 µM), we were able to determine the percentage of fast, mixed and slow component of the current (Burgard et al., 1999), that corresponds to homomeric P2X2, P2X3, or heteromeric P2X2/3 receptors. In TG neurons, we found that the fast component was evident in 56% of all tested cells, the mixed type could be observed in 44% of cells with the slow component not being detected in our sample (n=18). In DRG neurons (n=8), we found fast currents in 50% of cells and 50% of cells generated mixed currents, but no slow responses were observed. In contrast, in NDG neurons (n=11) we found 22% fast responses, no mixed and 78% slow replies (study I, Fig 6 A, B-D first row). Thus, rat TG and DRG neurons both express both homomeric and heteromeric P2X2/P2X3 subunits, whereas NDG neurons predominantly express heteromeric P2X2/P2X3 receptors.
Furthermore, our tests revealed that AppNHppA selectively depressed fast responses, with little or no effect on slow component of mixed currents. There was extensive inhibition of the fast desensitizing currents i.e. by 80.3±4.4% in TG, by 79.2±5.6% in DRG, but by only 16.8±11.8% in NDG neurons (study I, Fig 6 B-F, second row). The inhibition of the sustained currents was much weaker (by 15±6%, by 18±6%, and by 2±4% for TG, DRG and NDG,
respectively, study I, Fig 6 E-G). In general, data obtained in native sensory neurons were consistent with our results obtained from rat recombinant homomeric P2X receptors. These data further support the proposal that the inhibitory action by Ap4A-analogues is mediated via an HAD mechanism primarily on homomeric P2X3 receptors with little effect on heteromeric P2X2/3.
5.1.4 Ap4A-analogues induced effects on human P2X3 receptors
For translational purposes we explored the inhibitory and activation effects of AppNHppA and AppCH2ppA on recombinant homomeric human P2X3 (hP2X3) receptors. Importantly, the inhibitory potency of AppNHppA was confirmed on the hP2X3 receptor. Moreover, the inhibition by AppNHppA in the human homologue was even stronger than on the rat P2X3 receptor. Thus, 10 nM AppNHppA was able to decrease hP2X3-induced currents to 80±5%
of control, 100 nM AppNHppA inhibited them to 48±8% and 1 µM AppNHppA inhibited the response to 12±4% of the control values (study I, Fig 7, A-C). Interestingly, the partial agonist activity of the AppNHppA remained similar to that seen at rP2X3 receptors. Again, this required a concentration of AppNHppA exceeding 10 µM, i.e. ~1000 times higher than the concentration needed to evoke the inhibitory effects (study I, Fig 7 D). Similarly, the inhibitory effect of AppCH2ppA on hP2X3 receptors did not differ from inhibition on rP2X3 receptor (56±7%). Inhibited currents recovered after a wash-out time for both Ap4 A-analogues (I, Fig 7 E-F)
5.1.5 Analgesic effects of the Ap4A-analogues in vivo
As an additional approach, in collaboration with Ph.D. V. Viatchenko-Karpinsky (Ukraine), we assessed the anti-nociceptive effects of AppNHppA and AppCH2ppA using behavioral testing in vivo with inflammatory pain models. Thus, we injected diluted formalin into a rat’s hind paw, which evoked a biphasic nociceptive response. Immediate (acute phase) and tonic (chronic, inflammatory phase)(Coderre et al., 1990; Tjølsen et al., 1992). The acute phase of the formalin response developed at 0–5 min after injection and consisted of a direct effect of formalin on the nociceptive receptors. This first phase was followed by the tonic phase which developed within 7 – 45 min after injection and induced changes in animal behaviour, caused by hyperalgesia which develops due to a nociceptive and spinal neurons sensitization (O’Connor and Dworkin, 2009). Simultaneous injection of formalin with AppNHppA or AppCH2ppA into the hind paw of rats greatly decreased the number of formalin-induced nocifensive responses (i.e. responses evoked by activation of nociceptors). Next, we calculated the IC50 values for acute and chronic phases based upon the locally administered concentrations of AppNHppA and AppCH2ppA. The calculated IC50 values including effective in vivo IC50 values (mg/kg) are shown in Table 2. Post AppCH2ppA administration, nocifensive behavior was reduced by 60 times more effectively in the tonic phase compared with the acute phase of the formalin assay (study I, Fig 8). No increase in the nocifensive behavior was observed with control injections of Ap4A-analogues alone.
Table 2. IC50 values calculated from the formalin test Title IC50 in the acute
phase
IC50 in the tonic phase
effective IC50 in vivo
AppNHpp