tetrodotoxin and Peripheral-Nervous-System-Neoplasms

The novel sodium channel PN3/alpha-SNS, which was cloned from a rat dorsal root ganglion (DRG) cDNA library, is expressed predominantly in small sensory neurons and may contribute to the tetrodotoxin-resistant (TTXR) sodium current that is believed to be associated with central sensitization in chronic neuropathic pain states. To assess further the role of PN3, we have used electrophysiological, in situ hybridization and immunohistochemical methods to monitor changes in TTXR sodium current and the distribution of PN3 in normal and peripheral nerve-injured rats. (1) Whole-cell patch-clamp recordings showed that there were no significant changes in the TTXR and TTX-sensitive sodium current densities of small DRG neurons after chronic constriction injury (CCI) of the sciatic nerve. (2) Additionally, in situ hybridization showed that there was no change in the expression of PN3 mRNA in the DRG up to 14 d after CCI. PN3 mRNA was not detected in sections of brain and spinal cord taken from either normal or nerve-injured rats. (3) In contrast, immunohistochemical studies showed that major changes in the subcellular distribution of PN3 protein were caused by either CCI or complete transection of the sciatic nerve. The intensity of PN3 immunolabeling decreased in small DRG neurons and increased in sciatic nerve axons at the site of injury. The alteration in immunolabeling was attributed to translocation of presynthesized, intracellularly located PN3 protein from neuronal somata to peripheral axons, with subsequent accumulation at the site of injury. The specific subcellular redistribution of PN3 after peripheral nerve injury may be an important factor in establishing peripheral nerve hyperexcitability and resultant neuropathic pain.

Topics: Animals; Biological Transport; Drug Resistance; Immunohistochemistry; Male; Nerve Compression Syndromes; Nervous System Diseases; Neuroma; Neurons, Afferent; Patch-Clamp Techniques; Peripheral Nervous System Neoplasms; Rats; Rats, Sprague-Dawley; Reference Values; RNA, Messenger; Sciatic Nerve; Sodium Channels; Tetrodotoxin; Tissue Distribution

RT4-B is one of several cell lines derived from a multipotent stem cell line, RT4-AC, which originated from a rat peripheral neurotumor. Based on Northern blot and ribonuclease protection experiments, RT4-B8 cells have been proposed to express rat cardiac Na channel mRNA as the major isoform. We report here direct electrophysiological evidence that the expressed voltage-gated Na channels in the RT4-B8 cell line are of the cardiac phenotype with no evidence for subpopulations expressing other Na channel isoforms. Current activation half point (conductance) was -41 +/- 5 mV (n = 7) and the steady-state voltage-dependent availability half point was -89 +/- 1 mV. As expected for cardiac Na channels, the half concentration of block for tetrodotoxin block was 0.74 microM, for saxitoxin (STX) was 0.15 microM, and for the class 2B divalent cation Cd2+ was 67 microM. Block was well described by single-site dose-response relationships with no indication of a subpopulation with "neuronal" affinity. Single-channel conductance (140 mM Na+) was 10 pS and predicted the average number of channels open at peak Na current to be 3 channels/microns2. [3H]STX binding data were also consistent with a single population of low-affinity STX binding sites and predicted channel density to be 11 sites/microns2. No inwardly or outwardly rectifying K or Ca currents were detected electrophysiologically, although in some cells a small time-independent Cl current was detected. Reverse transcription-polymerase chain reaction of mRNA isolated from RT4-B8 cells demonstrated the presence of rat cardiac (rH1) and brain IIa alpha-subunit mRNA, as well as mRNA for the Na channel beta 1-subunit. Northern blot analysis confirmed the predominance of the rat cardiac Na mRNA compared with brain IIa. The beta 1-subunit mRNA levels were significantly lower than those detected in rat brain and heart mRNA but were comparable to the low level of beta 1-subunit mRNA detected in isolated rat ventricular myocytes.

Topics: Animals; Blotting, Northern; Electrophysiology; Myocardium; Peripheral Nervous System Neoplasms; Phenotype; Polymerase Chain Reaction; Rats; Saxitoxin; Sodium Channels; Transcription, Genetic; Tumor Cells, Cultured