Acid-sensing ion channel blocker diminazene facilitates proton-induced excitation of afferent nerves in a similar manner that Na+/H+ exchanger blockers do.

Tissue acidification causes sustained activation of primary nociceptors, which causes pain. In mammals, acid-sensing ion channels (ASICs) are the primary acid sensors; however, Na+/H+ exchangers (NHEs) and TRPV1 receptors also contribute to tissue acidification sensing. ASICs, NHEs, and TRPV1 receptors are found to be expressed in nociceptive nerve fibers. ASIC inhibitors reduce peripheral acid-induced hyperalgesia and suppress inflammatory pain. Also, it was shown that pharmacological inhibition of NHE1 promotes nociceptive behavior in acute pain models, whereas inhibition of TRPV1 receptors gives relief. The murine skin-nerve preparation was used in this study to assess the activation of native polymodal nociceptors by mild acidification (pH 6.1). We have found that diminazene, a well-known antagonist of ASICs did not suppress pH-induced activation of CMH-fibers at concentrations as high as 25 µM. Moreover, at 100 µM, it induces the potentiation of the fibers' response to acidic pH. At the same time, this concentration virtually completely inhibited ASIC currents in mouse dorsal root ganglia (DRG) neurons (IC50 = 17.0 ± 4.5 µM). Non-selective ASICs and NHEs inhibitor EIPA (5-(N-ethyl-N-isopropyl)amiloride) at 10 µM, as well as selective NHE1 inhibitor zoniporide at 0.5 µM induced qualitatively the same effects as 100 µM of diminazene. Our results indicate that excitation of afferent nerve terminals induced by mild acidification occurs mainly due to the NHE1, rather than acid-sensing ion channels. At high concentrations, diminazene acts as a weak blocker of the NHE. It lacks chemical similarity with amiloride, EIPA, and zoniporide, so it may represent a novel structural motif for the development of NHE antagonists. However, the effect of diminazene on the acid-induced excitation of primary nociceptors remains enigmatic and requires additional investigations.

Temperature increase significantly enhances nociceptive responses of C-fibers to ATP, high K+, and acidic pH in mice.

It is well established that temperature affects the functioning of almost all biomolecules and, consequently, all cellular functions. Here, we show how temperature variations within a physiological range affect primary afferents' spontaneous activity in response to chemical nociceptive stimulation. An ex vivo mouse hind limb skin-saphenous nerve preparation was used to study the temperature dependence of single C-mechanoheat (C-MH) fibers' spontaneous activity. Nociceptive fibers showed a basal spike frequency of 0.097 ± 0.013 Hz in control conditions (30°C). Non-surprisingly, this activity decreased at 20°C and increased at 40°C, showing moderate temperature dependence with Q10∼2.01. The fibers' conduction velocity was also temperature-dependent, with an apparent Q10 of 1.38. Both Q10 for spike frequency and conduction velocity were found to be in good correspondence with an apparent Q10 for ion channels gating. Then we examined the temperature dependence of nociceptor responses to high K+, ATP, and H+. Receptive fields of nociceptors were superfused with solutions containing 10.8 mM K+, 200 µM ATP, and H+ (pH 6.7) at three different temperatures: 20, 30, and 40°C. We found that at 30 and 20°C, all the examined fibers were sensitive to K+, but not to ATP or H+. At 20°C, only 53% of fibers were responsible for ATP; increasing the temperature to 40°C resulted in 100% of sensitive fibers. Moreover, at 20°C, all observed fibers were silent to pH, but at 40°C, this number was gradually increased to 87.9%. We have found that the temperature increase from 20 to 30°C significantly facilitated responses to ATP (Q10∼3.11) and H+ (Q10∼3.25), leaving high K+ virtually untouched (Q10∼1.88 vs. 2.01 in control conditions). These data suggest a possible role of P2X receptors in coding the intensity of non-noxious thermal stimuli.

Surface charge impact in low-magnesium model of seizure in rat hippocampus.

Putative mechanisms of induction and maintenance of seizure-like activity (SLA) in the low Mg(2+) model of seizures are: facilitation of NMDA receptors and decreased surface charge screening near voltage-gated channels. We have estimated the role of such screening in the early stages of SLA development at both physiological and room temperatures. External Ca(2+) and Mg(2+) promote a depolarization shift of the sodium channel voltage sensitivity; when examined in hippocampal pyramidal neurons, the effect of Ca(2+) was 1.4 times stronger than of Mg(2+). Removing Mg(2+) from the extracellular solution containing 2 mM Ca(2+) induced recurrent SLA in hippocampal CA1 pyramidal layer in 67% of slices. Reduction of [Ca(2+)](o) to 1 mM resulted in 100% appearance of recurrent SLA or continuous SLA. Both delay before seizure activity and the inter-SLA time were significantly reduced. Characteristics of seizures evoked in low Mg(2+)/1 mM Ca(2+)/3.5 K(+) were similar to those obtained in low Mg(2+)/2 Ca(2+)/5mM K(+), suggesting that reduction of [Ca(2+)](o) to 1 mM is identical to the increase in [K(+)](o) to 5 mM in terms of changes in cellular excitability and seizure threshold. An increase of [Ca(2+)](o) to 3 mM completely abolished SLA generation even in the presence of 5 mM [K(+)](o). A large variation in the ability of [Ca(2+)](o) to stop epileptic discharges in initial stage of SLA was found. Our results indicate that surface charge of the neuronal membrane plays a crucial role in the initiation of low Mg(2+)-induced seizures. Furthermore, our study suggests that Ca(2+) and Mg(2+), through screening of surface charge, have important anti-seizure and antiepileptic properties.