OBJECTIVETo observe the effects of chronic lead exposure on mRNA and protein expression of ASIC1a, ASIC2a, ASIC2b in hippocampus of baby-rats.

METHODSThe Wistar pregnant rats were randomly divided into 3 groups fed with distilled water or lead contained water (0.2% and 1.0% lead acetate) respectively, 5 rats in each group. The lead-exposure ranged from the 0 day of pregnancy to the offspring weaned. Then the baby-rats were fed with lead water like their mothers and killed at postnatal day 8 or 50. Atomic absorption spectrometry was used to determine lead content in the brain. RT-PCR and Western blotting were used to observe mRNA and protein expression of ASIC1a, ASIC2a and ASIC2b in their hippocampus respectively.

RESULTSThe brain lead content of test groups was higher than that of the control group (P < 0.01), and the lead content of the postnatal day 50 was higher than that in postnatal day 8 (P < 0.01). Compared with the control group, ASIC1a mRNA expression of 1.0% lead exposure in the hippocampus was uptrend (P < 0.01), ASIC1a protein expression of each test group was downtrend (P < 0.05), while for ASIC2a and ASIC2b mRNA and protein, there was no significant differences observed (P > 0.05).

CONCLUSIONASIC1a expression in hippocampus can be changed by chronic lead exposure.

Acid Sensing Ion Channels ; Animals ; Female ; Hippocampus ; drug effects ; metabolism ; Lead ; toxicity ; Nerve Tissue Proteins ; genetics ; metabolism ; Pregnancy ; Prenatal Exposure Delayed Effects ; genetics ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Sodium Channels ; genetics ; metabolism

OBJECTIVEThis study sought to isolate and identify the proteins that interact with ataxin-3, to confirm the interacted domain, and to provide new clues for exploring the function of ataxin-3 and the pathogenesis of spinocerebellar ataxia type 3 and Machado-Joseph disease (SCA3/MJD).

METHODSYeast two-hybrid screen (MATCHMAKER GAL4 Two-Hybrid System 3) and regular molecular biologic techniques were undertaken to screen human brain cDNA library with mutant ataxin-3 bait. Two baits from both normal and mutant C-terminus of ataxin-3 were created by subcloned methods to determine which domain of ataxin-3 interacts with the putative associated proteins and to find out optimal candidate proteins that interact with C-terminus of ataxin-3. Confocal microscope was used to observe whether ataxin-3 co-localized with the obtained interacting proteins in mammalian cells.

RESULTSFive novel ataxin-3 interacting proteins were obtained, among which were three known proteins, namely human rhodopsin guanosine diphosphate dissociation inhibitor alpha, small ubiquitin-like modifier 1, and human neuronal amiloride-sensitive cation channel 2; the other two were unknown. Interacting domain analysis revealed that an unknown protein interacted with the C-terminus near the polyglutamine tract of ataxin-3, the other four all interacted with the N-terminus. In the nucleus of SH-SY5Y cell, small ubiquitin-like modifier 1 co-localized with the wild-type ataxin-3 and with the intranuclear aggregates formed by the mutant ataxin-3.

CONCLUSIONAn unknown protein probably interacting with C-terminus of ataxin-3 is firstly discovered, and the initiative findings suggest first that the interaction of small ubiquitin-like modifier 1 with N-terminus of ataxin-3 and the relevant sumoylation probably participate in the post-translation modifying of ataxin-3 and in the pathogenesis of SCA3/MJD.

Acid Sensing Ion Channels ; Ataxin-3 ; Cell Line, Tumor ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Confocal ; Mutation ; Nerve Tissue Proteins ; genetics ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; Plasmids ; genetics ; Protein Binding ; Recombinant Fusion Proteins ; genetics ; metabolism ; Repressor Proteins ; genetics ; metabolism ; SUMO-1 Protein ; genetics ; metabolism ; Sodium Channels ; genetics ; metabolism ; Transfection ; Two-Hybrid System Techniques

This study aims to validate our hypothesis that acid-sensing ion channels (ASICs) may contribute to the symptom of pain in patients with chronic prostatitis (CP). We first established a CP rat model, then isolated the L5-S2 spinal dorsal horn neurons for further studies. ASIC1a was knocked down and its effects on the expression of neurogenic inflammation-related factors in the dorsal horn neurons of rat spinal cord were evaluated. The effect of ASIC1a on the Ca²⁺ ion concentration in the dorsal horn neurons of rat spinal cord was measured by the intracellular calcium ([Ca²⁺]i) intensity. The effect of ASIC1a on the p38/mitogen-activated protein kinase (MAPK) signaling pathway was also determined. ASIC1a was significantly upregulated in the CP rat model as compared with control rats. Acid-induced ASIC1a expression increased [Ca²⁺]i intensity in the dorsal horn neurons of rat spinal cord. ASIC1a also increased the levels of neurogenic inflammation-related factors and p-p38 expression in the acid-treated dorsal horn neurons. Notably, ASIC1a knockdown significantly decreased the expression of pro-inflammatory cytokines. Furthermore, the levels of p-p38 and pro-inflammatory cytokines in acid-treated dorsal horn neurons were significantly decreased in the presence of PcTx-1, BAPTA-AM, or SB203580. Our results showed that ASIC1a may contribute to the symptom of pain in patients with CP, at least partially, by regulating the p38/MAPK signaling pathway.
Acid Sensing Ion Channel Blockers/pharmacology* ; Acid Sensing Ion Channels/genetics* ; Animals ; Calcium/metabolism* ; Chelating Agents/pharmacology* ; Chronic Disease ; Cytokines/metabolism* ; Disease Models, Animal ; Egtazic Acid/pharmacology* ; Gene Knockdown Techniques ; Imidazoles/pharmacology* ; Inflammation/metabolism* ; MAP Kinase Signaling System/genetics* ; Male ; Pain/genetics* ; Peptides/pharmacology* ; Phosphorylation/drug effects* ; Posterior Horn Cells/metabolism* ; Prostatitis/complications* ; Protein Kinase Inhibitors/pharmacology* ; Pyridines/pharmacology* ; Rats ; Spider Venoms/pharmacology* ; Up-Regulation ; p38 Mitogen-Activated Protein Kinases/metabolism*

Amiloride and benzamil showed antinocicepitve effects in several pain models through the inhibition of acid sensing ion channels (ASICs). However, their role in neuropathic pain has not been investigated. In this study, we investigated the effect of the intrathecal amiloride and benzamil in neuropathic pain model, and also examined the role of ASICs on modulation of neuropathic pain. Neuropathic pain was induced by L4-5 spinal nerve ligation in male Sprague-Dawley rats weighing 100-120 g, and intrathecal catheterization was performed for drug administration. The effects of amiloride and benzamil were measured by the paw-withdrawal threshold to a mechanical stimulus using the up and down method. The expression of ASICs in the spinal cord dorsal horn was also analyzed by RT-PCR. Intrathecal amiloride and benzamil significantly increased the paw withdrawal threshold in spinal nerve-ligated rats (87%+/-12% and 76%+/-14%, P=0.007 and 0.012 vs vehicle, respectively). Spinal nerve ligation increased the expression of ASIC3 in the spinal cord dorsal horn (P=0.01), and this increase was inhibited by both amiloride and benzamil (P<0.001 in both). In conclusion, intrathecal amiloride and benzamil display antinociceptive effects in the rat spinal nerve ligation model suggesting they may present an alternative pharmacological tool in the management of neuropathic pain at the spinal level.
Acid Sensing Ion Channels/genetics/metabolism ; Amiloride/*analogs & derivatives/pharmacology/*therapeutic use ; Analgesics/pharmacology/*therapeutic use ; Animals ; Disease Models, Animal ; Male ; Neuralgia/*drug therapy ; RNA, Messenger/metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Spinal Cord/metabolism ; Transcription, Genetic/drug effects