The endogenous redox agent L-cysteine induces T-type Ca2+ channel-dependent sensitization of a novel subpopulation of rat peripheral nociceptors
ABSTRACT Recent studies have demonstrated a previously unrecognized contribution of T-type Ca2+ channels in peripheral sensory neurons to pain sensation (nociception). However, the cellular mechanisms underlying the functions of these channels in nociception are not known. Here, in both acutely dissociated and intact rat dorsal root ganglion neurons, we characterize a novel subpopulation of capsaicin- and isolectin B4-positive nociceptors that also expresses a high density of T-type Ca2+ currents. Using these "T-rich" cells as a model, we demonstrate that the endogenous reducing agent L-cysteine lowers the threshold for nociceptor excitability and induces burst firing by increasing the amplitude of T-type currents and shifting the gating parameters of T-type channels. These findings, which provide the first direct evidence of T-type Ca2+ channel involvement in the control of nociceptor excitability, suggest that endogenous T-type channel agonists may sensitize a unique subpopulation of peripheral nociceptors, consequently influencing pain processing under normal or pathological conditions.
- SourceAvailable from: Slobodan M Todorovic
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- "Acutely isolated DRG neurons DRG cells from adolescent rats were prepared as previously described (Todorovic et al. 1998; Nelson et al. 2005; Choe et al. 2011). For recording, cells were plated onto uncoated glass coverslips, placed in a culture dish, and perfused with external solution. "
ABSTRACT: T-type calcium channels (T-channels) play an important role in controlling excitability of nociceptors. We have previously shown that a synthetic series of 5β-reduced steroids induce a voltage-dependent blockade of T-currents in rat dorsal root ganglia (DRG) cells in vitro and induce potent analgesia to thermal stimuli in rats in vivo (Mol Pharmacol 66:1223-1235, 2004). Here, we investigated the effects of the endogenous 5β-reduced neuroactive steroid molecule, epipregnanolone [(3β,5β)-3-hydroxypregnan-20-one], on peripheral nociception. We used acutely dissociated DRG cells in vitro from adult rats as well as in vivo pain studies in mice and rats to investigate the effects of epipregnanolone on DRG T-channels. We found that epipregnanolone reversibly blocked DRG T-currents with a half-maximal inhibitory concentration (IC50) of 2 μM and stabilized the channel in the inactive state. However, sodium, potassium, and gamma-aminobutyric acid (GABA)-gated ionic currents were not sensitive to the blocking effects of epipregnanolone even at 10 μM. In ensuing in vivo studies, we found that intraplantar (i.pl.) injections of epipregnanolone directly into peripheral receptive fields reduced responses to nociceptive heat stimuli in rats in a dose-dependent fashion. Furthermore, i.pl. epipregnanolone injections effectively reduced responses to peripheral nociceptive thermal and mechanical stimuli in wild-type mice but had no effect on the responses of CaV3.2 knockout mice. We conclude that the inhibition of peripheral CaV3.2 T-channels contributes to the potent analgesic effect of the endogenous steroid epipregnanolone.Psychopharmacology 05/2014; 231(17). DOI:10.1007/s00213-014-3588-0 · 3.99 Impact Factor
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- "In order to corroborate recombinant and native channel pharmacology , whole cell calcium currents were recorded from medium sized dissociated mouse DRG neurons expressing low voltage–activated T-type currents of large amplitude as well as high-voltage-activated (HVA) currents   . It is well established that T-type currents in somatic medium and small sized rat and mouse DRGs exhibit Ca V 3.2-like properties including the pharmacological inhibition by low concentrations of nickel ions      , corroborating the prevalent expression of the Ca V 3.2 transcript and protein in these neurons    . "
ABSTRACT: T-type calcium channels encoded by the Ca(V)3.2 isoform are expressed in nociceptive primary afferent neurons where they contribute to hyperalgesia and thus are considered as a potential therapeutic target to treat pathological pain. Here we report that the small organic state-dependent T-type channel antagonist TTA-A2 efficiently inhibits recombinant and native Ca(V)3.2 currents. Although TTA-A2 is a pan Ca(V)3 blocker, it demonstrates a higher potency for Ca(V)3.2 compared to Ca(V)3.1. TTA-A2 selectivity for T-type currents was demonstrated in sensory neurons where it lowered cell excitability uniquely on neurons expressing T-type channels. In vivo pharmacology in Ca(V)3.2 knockout and wild type mice reveal that TTA-A2-mediated antinociception critically depends on Ca(V)3.2 expression. The pathophysiology of irritable bowel syndrome (IBS) was recently demonstrated to involve Ca(V)3.2 in a rat model of this disease. Oral administration of TTA-A2 produced a dose-dependent reduction of hypersensitivity in an IBS model, demonstrating its therapeutic potential for the treatment of pathological pain. Overall, our results suggest that the high potency of TTA-A2 in the depolarized state strengthen its analgesic efficacy and selectivity toward pathological pain syndromes. This characteristic would be beneficial for the development of analgesics targeting T-type channels, in particular for the treatment of pain associated with IBS.Pain 11/2012; 154(2). DOI:10.1016/j.pain.2012.10.023 · 5.84 Impact Factor
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- "Precise relationship between Ca V 2.3 and Ca V 3.2 channels in modulating nociceptive transmission in DH neurons is not known but it is possible that Ca V 3.2 channels play a permissive role. It has been shown that T-currents have a small fraction of channels that are active at physiological membrane potentials contributing to " window current " that can tonically depolarize neurons (Perez-Reyes, 2003; Nelson et al., 2005). Thus, we propose that presynaptic Ca V 3.2 channels may be involved in setting a relatively depolarized resting membrane potential that could in turn allow Ca V 2.3 channels to drive secretion in nociceptive DH neurons. "
ABSTRACT: It is generally accepted that presynaptic transmitter release is mainly regulated by subtypes of neuronal high-voltage-activated Ca(2+) channels. Here for the first time, we examined the role of T-type Ca(2+) channels (T-channels) in synaptic transmission in the dorsal horn (DH) of the spinal cord using patch-clamp recordings from acute spinal cord preparations from both rat and mouse. We found that selective pharmacological antagonism of T-channels inhibited spontaneous synaptic release of glutamate in superficial laminae I-II of the DH, while GABA release was spared. We found similar effect in identified nociceptive projection neurons of lamina I of the DH, but not in inhibitory DH interneurons. In comparison, antagonism of T-channels did not affect excitatory transmission in deeper non-nociceptive DH laminae. Furthermore, we used isoform-specific agents, knock-out mice and immunohistochemistry to specifically implicate presynaptic Ca(V)3.2 channels. We also used an animal model of painful diabetic neuropathy to demonstrate that blocking T-channels in superficial DH neurons suppressed spontaneous excitatory synaptic transmission in diabetic rats in greater degree than in healthy age-matched animals. These studies provide previously unknown information regarding the role of presynaptic T-channels in nociceptive signaling in the spinal cord.The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 07/2012; 32(27):9374-82. DOI:10.1523/JNEUROSCI.0068-12.2012 · 6.75 Impact Factor