Temporal-spatial Expression Of Gap Junctional Cennexins And Their Phosphorylation Modulating Mirror Image Pain

Nowadays, pain is an intractable biomedical scientific and clinical problem to human health. Pain could be induced by many kinds of reasons including tissue injury, inflammation, emotion-dysfunction, sensorimotor disorders, genetic mutations, cancer and so on. Persistence, paroxysmal, chronicalization, diversities,personalization of pain profiles are involved in detection, transmission, synaptic integration of noxious stimuli for individual and adaptive plasticity. It thus leads the occurrence of spontaneous pain, primary and secondary, and also mirror image pain hypersensitivity. However, the understanding of pain mechanisms and control, especially of mirror image pain, is still far from preventation, diagnosis, translational medical treatment, although more and more rapid development of pain researches and controls,novel discovery of anti-pain drugs and strategies has been found.Accumulative evidences suggested that gap junctional connexin channels or hemiochannels between glia-neuronal intercellular communications expressed diverse cellular specific subtypes, distribution, phosphorylation, and modulated pain. In this paper, based on our well-established the experimental animal mirror image pain model mimicking natural scorpion sting inducing pain, the single side of rat hindpaw plantar subcutaneous injection of specific sodium channel activator Bm K I, one of main algogenic peptidergic components from the venom of Chinese scorpion Buthus Martensi Karsch(Bm K), was applied as pain-evoking stimulus. Combining with behavioral observation, real-time quantitative PCR, immunoblotting, immunofluorescent, living cell image and other techniques, the dynamic temporal-spatial expression in peripheral and central cellular connexins and their phosphorylations were investigated. The main research contexts and results were as follows:i. The distinct expression and distribution of connexin subtypes in spinal cord and dorsal root ganglionqRT-PCR screening showed that the connexin subtypes in rat spinal cord and dorsal root ganglion(DRG) was quite different. It was found that spinal connexins mainly include Cx47、Cx45、Cx43、Cx36、Cx32、Cx30、Cx29 and Cx26, and those in DRG are Cx45、Cx43、Cx39、Cx37、Cx36、Cx32、Cx31.1、Cx31、Cx30、Cx29 and Cx26.Moreover, as for the same subtype, the expression density is divergent in spinal cord and dorsal root ganglion. The results suggest a cellular differentiation of expression and functional modulation of connexin subtypes.ii. The open status and quantitative changes of connexin subtypes modulating mirror image painBm K I-induced behaviors were significantly by pre-treatment intrathecal injection of gap junctional channel inhibitor CBX、GA and Cx 43 mimetic peptide Gap26 in a dose-dependent manner, suggesting that the opened gap junctional channels participate in the induction, development and maintenance of pain. The further quantitative assays showed that the amount of connexin subtypes changes with different “time windows” in bilateral spinal cord and DRG during pain development. It meant that, in spinal cord, the changes of Cx 36 were “n” shaped both at bilateral sides, Cx 43 were “v” shaped, and however, Cx 32 were “n” shaped in ipsilateral and “v” shaped in contralateral side;contrast in DRG, the expression of all of three subtypes of connexins were increased earlierly, and then decreased latterly. Most of important, the expression changes of spinal and DRG cennnexins could significantly reversed by pre-treatment with intrathecal CBX at distinct degrees. These results suggest that the functional open and close control, and expression quantitative changes of connenxin channels, especially Cx 43, regulate the mirror image pain.iii. The co-localization of connexin subtypes and mirror image painIn spinal dorsal horn, Cx 32 was co-localized evenly with neuronal marker Neu N,astrocyte GFAP and microglia Iba1, and Cx 36 distributed at deeper laminar co-localizing mainly with Neu N, but few with GFAP and Iba1, and Cx 43 co-localized with GFAP, but few with Iba1 and none with Neu N. In DRG, Cx 32, Cx 36 or Cx 43 could co-localize with NF200, a protein marker of large-diameter neurons. And, Cx 32 expressed in IB4positive neurons, but not in CGRP neurons. However, Cx 36 and Cx 43 were only found in GFAP and Iba1 positive satellite glia. Furthermore, the both rates of co-localized expression of cennexins were reduced by pre-treatment intrathecal injection of CBX.These results indicate that the specific distribution and co-localization of connexins also play critical roles in mirror image pain, and the subtypes of connexins could be well-used as cellular marker proteins or convergent sensors for pain discrimination.iv. Phosphorylation modification of Cx 43 and mirror image painWestern blotting assays showed that Cx 43 phosphorylation changed drastically both in spinal cord and DRG under pain stimuli. In spinal dorsal horn, at ipsilaeral side to Bm K I injection, the expression change of p Cx43(Ser262) was “v” shaped,p Cx43(Tyr265) was “n” shaped, and p Cx43(Ser279/282) and p Cx43(Ser368) was increased gradually; however, at contralateral side, all phosphorylation sites only manifested increase patterns. In DRG, the detectable p Cx43(Ser368) was “n” shaped at ipsilateral side, but just decreased at contralateral side. Corresponding to expression pattern of Cx 43 protein, these results further support that phosphorylation modification of connexin subtpyes, such as Cx 43, is relative to gating, transcription, translation and post-translational trafficking of connnexins, by which to meet the spinal and DRG control of mirror image pain.Finally, it concludes that the dynamic and differential temporal-spatial expression of connnexin subtypes, especially Cx 43 if any more, asymmetrical at both side to noxious stimuli, is an peripheral and central endogenous control and modulation mechanisms of mirror image pain.