| T-type Ca2+ channels(also named as the low-voltage active calcium channels, LVA), consists of three subunits, Cav3.1, Cav3.2, Cav3.3, encoded by CACNA1 G, CACNA1 H and CACNA1 I genes, respectively. These channels have the property of low threshold for activation, thus are partially active at voltages near the neuronal resting membrane potential; they also have the characteristics of fast activation, fast inactivation, and slow deactivation kinetics. More and more investigations show that T-type Ca2+ channels play an important role in peripheral pain pathways. In the peripheral somatosensory system, T-type Ca2+ channels express in the small, TRPV1-positive nociceptive neurons and in two populations of low-threshold mechanoreceptors(LTMRs): Aδ- and C-LTMRs, which innervate skin hair follicles. Cav3.2 is the predominant T-type channel isoform expressed in sensory neurons. Cav3.2 expression is reported in various compartments of peripheral fibers including peripheral nociceptive nerve endings and axons of skin afferents, nodes of Ranvier of Aδ fibers and pre-synaptic terminals of nociceptive fibers in the spinal cord. Conditional knock-out of Cav3.2 or specific knock-down of this subunit in dorsal root ganglion(DRG), using intrathecal injection of antisense oligonucleotides, resulted in potent anti-nociceptive effects in models of neuropathic and inflammatory pain. Pharmacological inhibitors of T-type Ca2+ channels consistently display analgesic efficacy in rodent pain models. Cav3 s are clinically validated drug targets for pain and several novel selective T-type channel blockers are currently under clinical trials as analgesics.A flip side of Cav3.2 expression in pain pathways is that an increase in this channel activity and/or abundance can have a proalgesic effect. T-typeCa2+ currents are often increased in pathological pain conditions, such as diabetic neuropathy, peripheral nerve injury or inflammation. Bradykinin(BK), adenosine triphosphate(ATP), norepinephrine bitartrate(NE) and prostaglandin E2(PGE2) are well known as inflammatory mediators which can cause pain, but until now there still no any systematic studies on their effect on T-type Ca2+ channel. In the first part of this thesis, we will study the effects and the underlying mechanism of these four inflammatory mediators on T-type Ca2+ channel in DRG neurons.Substance P(SP) is an eleven-amino acid neuropeptide of a tachykinin family which is produced in both peripheral and central nervous systems. SP is a major excitatory neuromodulator that is involved in pain transmission and smooth muscle contraction, and modulates inflammatory and immune responses. SP is abundantly produced by a subset of TRPV1-positive, pain-sensing(nociceptive) neurons of the mammalian peripheral somatosensory system. These neurons release SP in response to noxious stimulation both centrally and peripherally. Peripheral SP released from nociceptive nerve endings causes so-called ‘neurogenic inflammation’ while in the spinal cord SP functions as an excitatory co-transmitter alongside glutamate. Intracellular effects of SP are mediated by the G protein coupled neurokinin(NK1-3) receptors. No studies on the effect of substance P on T-type Ca2+ channel have been reported.Accumulating evidence show that T-type Ca2+ channel can be modulated by redox mechanism. L-cysteine or other reducing agents can significantly increase the T-type Ca2+ current in DRG neurons. Ascorbic acid and N2 O increase the release of ROS(reactive oxygen species) of cells, which can inhibit the T-type Ca2+ currents in DRG neurons. RNS(reactive nitrogen species) also modulate the function of T-type Ca2+ channel through redox mechanism. However, up to now, the mechanism for modulation of T-type Ca2+ channel by redox is still not clear. In the second part of this thesis we will investigate the effect of SP on T-type Ca2+ current on DRG neurons and Cav3 s currents in HEK cells and the related mechanism.The strong excitatory role of SP in spinal nociceptive pathways has triggered a massive, industry-wide quest for new analgesics based on NK1 receptor antagonists. However, despite some strong pre-clinical data, clinical trials of NK1 receptor antagonists failed. The reasons for the lack of analgesic efficacy of the systemically-applied NK antagonists are still unclear. However, one hypothesis is that, while excitatory in CNS(central nervous system), SP may exert paradoxical inhibitory/analgesic effects in the peripheral nociceptive pathways. In the third part of this thesis we will investigate the possible involvement of T-type Ca2+ channel in SP-mediated paradoxical inhibitory/analgesic effects. We will study the effect of S9SP(an agonist of NK1 receptor), Z944(selective inhibitor of T-type Ca2+ channel) and ST101(selective opener of T-type Ca2+ channel) on the excitability of DRG neurons, and on the pain behavior of animal pain models.Part 1 Effect of endogenous inflammatory mediators(BK, ATP,NE, PGE2) on T-type Ca2+ channel in DRG neurons.Objective: To investigate the modulation of T-type Ca2+ channel by endogenous inflammatory mediators in rat DRG neurons and the mechanism underlying the effects.Methods: 1 DRG neurons were acutely disassociated and were incubated with inflammatory agents overnight. 2 Whole-cell patch clamp,cell inmmunohistochemistry and RT-PCR were used to investigate the modulation of T-type Ca2+ channel and the mechanism of the modulation.Results: 1 Overnight treatment with the inflammatory cocktail of BK, ATP,NE and PGE2 significantly increased the percentage of the T-type-positive neurons from rat DRG from 21/43(48.8%) to 31/42(73.8%)(P < 0.05), but did not affect the peak T-type Ca2+ current amplitude; the mean T-type Ca2+ current amplitudes were-113.6 ± 18.5 p A and-105.3 ± 14.5 p A in the control and the inflammatory agents-treated neurons, respectively. During 24 h live-cell imaging of DRG cultures we did not observe any significant cell death induced by the inflammatory cocktail, as compared with the control. 2 Overnight incubation with BK or ATP significantly increased the percentageof T-type-positive neurons from 25/54(46.3%; control) to 43/54(79.6%; BK)(P < 0.05) or 39/56(69.6%; ATP)(P < 0.05), respectively. Neither NE nor PGE2 had an effect on the percentage of T-type-positive neurons. All the four compounds had no effect on T-type Ca2+ current amplitude. 3 PLC inhibitor U-73122 or B2 R blocker Hoe-140 completely abolished the effect of the inflammatory cocktail on the abundance of T-type-positive neurons in DRG culture. However, P2X2/P2X3 receptor antagonist A-317491 did not affect the increase in the proportion of T-type-positive neurons induced by the inflammatory treatment. 4 The proteasome inhibitor(MG132), which reduces the degradation of ubiquitin-conjugated proteins, had no effect on the proportion of T-type-positive neurons. RT-PCR result shown there was no significant increase of Cav3.2 transcript in DRG cultures after overnight incubation in the presence of the inflammatory cocktail. Confocal imaging of cultured DRG neurons immunostained for Cav3.2 revealed there were also no significant changes in the total Cav3.2 protein abundance in DRG neurons after either of inflammatory treatments(inflammatory cocktail or individual inflammatory mediators).Conclusion: 1 BK and/or ATP incubation increase proportion of T-type-positive DRG neurons. 2 The effect of BK and ATP is mediated by Gq/11-PLC pathway.Part 2 Substance P modulation of T-type Ca2+ channel of DRG neurons and the underlying mechanismObjective: To investigate the effect and the mechanism for SP modulation of T-type Ca2+ currents in rat DRG neurons.Methods: 1 Patch clamp technique was used to investigate the effect of SP on T-type Ca2+ currents in DRG neurons and in HEK293 cells. 2 si RNA gene silencing and pharmacology modulators were used to study the involvement of G protein-coupled receptor pathway, the redox and zinc in the effect of SP. 3 Mutagenesis study was used to study the action sites of SP on Cav3.2 channels.Results: 1 S9SP(an agonist of NK1 receptor) concentration-dependentlyinhibited T-type Ca2+ currents in DRG neurons, with an IC50 of 6.20 ± 2.99 n M. 2 1 μM S9 SP induced a maximal 22.5 ± 2.3% inhibition of the original current amplitude. Sar9-Met(O2)11-SP(another NK1 receptor agonist)(1 μM) also induced a similar(26.6 ± 4.7%) inhibition. On the other hand, the antagonist of NK1 receptor CP-99994 completely abolished the inhibition induced by S9 SP. 3 The S9SP-indcued inhibition of T-type Ca2+ currents was also completely abolished by pertussis toxin(PTX, a Gi/o protein inhibitor). A 80% knockdown of Gq/11 m RNA level(to the 20% of the basal level which was based on Gq/11 expression level measured in the scrambled si RNA-transfected neurons) by si RNA had no effect on the S9SP-induced inhibition of T-type Ca2+ currents(1 μM S9 SP inhibited T-type Ca2+ currents by 27.1 ± 3.8%). However, knockdown of Gi/o almost completely abolished the S9SP-induced inhibition of T-type Ca2+ currents(1 μM S9 SP inhibited T-type Ca2+ currents by 2.5 ± 2.6%). PKC inhibitor(BIM I) did not affect the effect of S9 SP. 4 DL-Dithiothreitol(DTT), a reducing agent, when applied after the S9SP-induced inhibition of T-type Ca2+ currents, completely reversed the inhibition. On the other hand, oxidative agents H2O2 and antimycin A mimicked the effect S9 SP and inhibited the T-type Ca2+ currents. 5 Application of 750 n M Zn Cl2 caused inhibition of T-type Ca2+ currents by ~25%, which also prevented a further inhibition by S9 SP. Furthermore, S9 SP did not produce any effect in the presence of the zinc chelator N,N,N’,N’-Tetrakis(2-pyridylmethyl)ethylenediamine(TPEN). S9SP-induced inhibition was completely reversed by bath application of TPEN(still in the presence of S9SP). 6 Zinc imaging and atomic absorption experiments show there was no detectable changes in either extracellular or intracellular zinc content after incubation of DRG with S9 SP. 7 S9 SP treatment strongly increased the efficacy of zinc-mediated inhibition via the high affinity mechanism while having no effect on the inhibition at higher concentration. 8 Mutagenesis results show high-affinity zinc binding site is necessary for Cav3 channel sensitivity to S9 SP. 9 MTSES(membrane impermeable oxidizing agent) but not NEM(membrane permeable oxidizing agent) applied inextracellular bath-solution mimicked S9SP’s effect on Cav3.2 currents overexpressed in HEK cells. 10 Mutation of three cysteines in S1-S2 linker and one in transmembrane region of S1 in repeat I completely abolished the inhibition induced by MTSES.Conclusion: 1 S9 SP, through NK1 receptor, activates Gi/o pathway, induces inhibition of T-type Ca2+ currents in DRG neurons. 2 S9 SP inhibits T-type Ca2+ currents with a ROS mechanism. 3 ROS modulates the affinity of T-type Ca2+ channel to zinc resulting in the inhibition of T-type Ca2+ currents. 4 One or several cysteines in the linker of S1-S2 in repeat I of Cav3.2 are involved in the redox reaction of Cav3.2 channel.Part 3 The role of SP-induced inhibition of T-type Ca2+ channel in neuronal excitability and peripheral analgesic effectObjective: To investigate the effect of SP-mediated modulation of T-type Ca2+ channel on the rat DRG neuronal excitability and on the pain behavior of animal models.Methods: 1 Current clamp was used to investigate the effect of Z944(selectively T-type Ca2+ channel inhibitor), S9SP(an agonist of NK1 receptor) and ST101(selectively T-type Ca2+ channel opener) on the excitability of cultured rat DRG neurons. 2 The pain behavior of rats was induced by injection of bradykinin(BK), mechanical and heat stimulation of rat hind paws; Z944, S9 SP and ST101 were injected into the hind paw of rats to study their effect on the pain behavior. 3 Antisense oligonucleotides against Cav3.2 were injected intrathecally in DRG(L4-L6) to lower down the expression Cav3.2.Results: 1 Z944 significantly increased the rheobase, decreased the action potential(AP) firing and hyperpolarized the cell membrane of the small-diameter DRG neurons without significantly affecting the AP amplitude or duration. In contrast, ST101 significantly increased the AP firing and depolarized the cell membrane potential(Erest) of DRG neurons; as in the case of Z944, the AP amplitude or duration was not affected. In further current clamp experiments application of S9 SP generally produced a pattern of effectssimilar to that of Z944 although only the effect on the rheobase reached significance. In si RNA gene silencing experiments, knocked-down of Cav3.2 expression in DRG neurons prevented Z994 and ST101 from producing any significant effect on the excitability of DRG neurons. 2 BK expectedly and significantly reduced the rheobase, increased the AP number and depolarised the Erest. When co-applied with BK, both SP and Z944 completely reversed the effects of BK on the rheobase and the AP number. 3 Hind-paw injection of 0.02 μM, 0.2 μM, 2 μM and 200 μM ST101 respectively, produced significantly nocifensive behavior. 4 Hind-paw injection of Z944(1 μM, 10 μM, 100 μM, respectively) significantly increased paw withdrawal latency upon exposure to radiant heat suggesting decreased sensitivity. 5 Hind-paw injection of Z944 significantly reduced the sensitivity to gentle touch. S9 SP injection induced similar effect. In contrast, injection of ST101 greatly increased response rates within the light touch range. 6 Pre-injection of both RTG(an opener of M-type K+ channel) and Z944 significantly attenuated the BK-induced pain. Pre-injection of S9 SP also strongly suppressed the BK-induced nocifensive behavior. Pre-injection of a mixture of RTG and Z944 was as efficacious as SP in reducing BK-induced nocifensive behavior and showed a tendency to be more efficacious as compared to RTG or Z944 injected alone. 7 Intrathecal injection of antisense oligonucleotides against Cav3.2 abolished the analgesic effect of Z944, and reduced the effect of S9 SP suggesting an involvement of T-type Ca2+ channels.Conclusion: 1 ST101 increases the excitability but Z944 and S9 SP reduce the excitability of the cultured rat DRG neurons. The effects of ST101 and Z944 are through the modulation on T-type Ca2+ channel. BK induces an increase in neuronal excitability of rat DRG which are reversed by Z944 and S9 SP. 2 ST101 induces pain; Z944 and S9 SP reverse the pain induced by BK. 3 Cav3.2 is fully responsible for Z944 induced analgesic effect and is partially responsible for SP-induced analgesic effect. |
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