In this study, we perform a microarray screening for genes with different expression in rat models of inflammatory visceral pain and demonstrate that Cav1.2 and Cav2.3 participates in the visceral hyperalgesia induced by TNBS-colitis. The result revealed that the level of Cav1.2 and Cav2.3 protein in L6-S2 DRG were significantly up-regulated following colitis and L-type and R-type calcium currents significantly increased in colonic specific DRG neurons, while visceral sensitivity to mechanical stimuli also increased significantly. Intrathecal administration of the specifically inhibits nimodipine and SNX482 reduced the colonic hyperalgesia to balloon distention.
Genes regulated in the L6-S2 DRG with TNBS treated may contribute to a variety of biologic processes such as inflammatory visceral pain signaling. In this study, we found that a large number of ion channels strongly regulated in DRG after TNBS treated such as VGCC (Cav1.2, Cav2.3), inositol 1,4,5-trisphosphate receptor 2, ryanodine receptors 1(RyR1), P2X7, P2Y and so on. Some studies have reported about the role of these channel in pain sensation such as RyR1  P2X7 [8–13] and P2Y1 . But these channels haven’t been studied in visceral sensation. In addition, P2X7 and P2Y1 receptors involved in the neuronal soma-satellite cell interaction [13, 15]. Maybe the up-regulation of P2X7 and P2Y1 can improve the neuron–glia communication. We also found that a large number of neurotransmitter and receptor strongly regulated in DRG after TNBS treated such as tumor necrosis factor (TNF, member 2 and 4), PGER(EP2), 5-HTR 1A, 5-HTR 2A and FGFR3, which upregulated in DRG of TNBS treated rat. Recently, several studies provided evidence that TNF-α may act on primary afferent neurons and induces hypersensitivity [12, 16, 17]. Whether the up-regulation of EP2 in L6-S2 DRGs of TNBS treated rats, mediating the visceral hypersensitivity, needed further study. The previous studies have suggested that 5-HT1A, 5-HT2A, 5-HT2C, 5-HT3 and 5-HT4 receptor subtypes in the DRG play important roles in facilitating peripheral nociceptive transmission [18–20]. Besides the ion channels and receptors, signal transduction molecules were also significantly changed. These changes could be associated with the rearrangement of pain sensory pathways that take place during the development of visceral inflammatory pain. All these changed genes maybe establish a positive feedback loop between neuron-neuron and neuron-satellite cell, thus contributing to exaggerated responses under pathological conditions which still need further experimental research proof.
In our study, we found that both the expression and the function of L- and R- type VGCC were upregulated in colonic DRG neurons of TNBS treated rats compare with control group. Kania has reported L-type VGCC blockers (nifedipine, diltiazem verapamil) can be useful in controlling acute visceral pain in the model of visceral pain or hypersensitivity of sheeps, which suggests that L-type VGCC play a crucial role in the modulation of acute visceral hyperalgesia [21–23]. They only use the pharmacology method in the behavioral test lacking the studies of molecular mechanism. In addition to visceral pain, neuronal L-type VGCCs Cav1.2 and Cav1.3 have reported to be up- or down regulated in the model of neuropathic pain in rats [24, 25]. R-type Ca2+ channels located at primary synapses  and can contribute to neurotransmitter release . R-type VGCCs are associated with nociceptive processing in various pain conditions. Mathews reported that spinal SNX-482, an R-type VGCC blocker, inhibited noxious C-fiber and Aδ-fiber-mediated dorsal horn neuronal responses in conditions of neuropathy, not in sham operated rats and that non-noxious Aδ-mediated responses did not affect by SNX-482 . In another study, mice lacking the α1E Ca2+ channel subunit exhibited normal pain behavior against acute mechanical, thermal, and chemical stimuli; however, they showed reduced responses to somatic inflammatory pain . So far, there has been no definitive study that shows that the role of R-type VGCC in visceral sensation. Our results revealed that upregulated Cav1.2 (L-type) and Cav2.3 (R-type) maybe involved in the inflammatory visceral hypersensitivity induced by TNBS treated.
The VGCCs manage and adjust neurotransmitter release and Ca2+-dependent depolarization that contribute to the characteristic firing patterns of most neurons, including those in pain pathways. As far, the role of VGCC in pain was not consensus. Because a change in [Ca2+]i can affect neuronal properties via changes in ion channel activity [30, 31], enzymatic activity  and/or gene expression , the physiological impact of the inflammation induced changes in evoked increases in [Ca2+]i will depend on where in the neuron they are manifest. On the other hand, an increase in [Ca2+]i close to functional Ca2+ dependent K+ channels should increase K+ channel activity resulting in a decrease in afferent excitability and the transmission of nociceptive information, while a change in the dynamics of Ca2+ signaling in the cell body may result in changes in gene expression that are either pro- or anti-nociceptive . Thus, the colon inflammation-induced changes in the regulation of [Ca2+]i may contribute to the visceral inflammatory hyperalgesia or serve as a feedback inhibitory mechanism functioning as a “break” to a host of pro-nociceptive inflammatory processes. We further detected the visceral pain after intrathecal injection of specific calcium channel inhibitors. Nimodipine specifically inhibits Cav1.2 α1C and to a lesser extent T-type VGCC in central and peripheral neurons. But the concentration for T-type Ca2+ channel inhibition is two orders of magnitude higher than for L-type Ca2+ channel . The SNX-482 is specific inhibitor for Cav2.3. Cav1 (L-type) can be partially inhibited by saturating concentrations of SNX-482 . SNX-482 is a large peptide and the concentration of SNX-482 applied was 5 μg/Kg. The concentration in the tissue were limited and this concentration can’t inhibit the Cav1 . As the intrathecal injection of nimodipine or SNX-482 significantly reduced the visceral inflammatory hyperalgesia in this study, we supposed that Cav1.2 and Cav2.3 may contribute to the visceral inflammatory hypersensitivity. However, additional experiments will be needed to study the direct consequence of the increase of [Ca2+]i on function of the sensory neurons.
In summary, our data revealed that there are many genes marked changed in L6-S2 DRGs of visceral inflammatory model, which encompass a large number of distinct family members including neuropeptides, receptors, ion channels, signal transduction molecules and others. Among these changed genes, the up-regulation of Cav1.2 and Cav2.3 contributed to visceral inflammatory hyperalgesia, which maybe the potential therapeutic targets for visceral pain.