| I. Pharmacological characteristics of desensitized nAChRs Nicotinic acetylcholine receptors (nAChRs) desensitization can be described as a decline or loss in response to agonist after repetitive or chronic exposure to agonists. Traditionally, nAChRs desensitization is viewed as a non-functional state. However, our previous studies indicated that desensitized nAChRs only were not mediating N-like response to agonist, indeed they were not a non-functional states and had specific physiological functions. Based on the viewpoint of nervous network, we observed modulations by desensitized nAChRs on gene expressions, protein functions and behaviors of animals, in order to further demonstrate the effects of desensitized nAChRs on brain functions and investigate the important roles of desensitized nAChRs involved in nicotine tolerence and addition.1. Modulation by desensitized nAChRs on expression profile of genes related to neurobiology in brainRats were treated with nicotine at the doses of 2.4mg/kg sc. twice a day for 14 days. RNA was extracted from the whole brain samples and converted to double-stranded cDNA and then to biotinylated cRNA. The biotinylated cRNA was fragmented, and hybridized to GeneChip (Affymetrix Rat Neurobiology U34). The chips were scanned with a probe array scanner, and the data were analyzed by the Affymetrix Microarray Analysis Suite (MAS), and were confirmed using RT-PCR. The results showed: expression of 224 transcripts altered, including 115 increased and 104 decreased compared with control groups. The major classes of transcripts altered were membrane receptors, kinases and channels (94 transcripts altered). In the neurotransmitter receptor system, there were 9 transcripts up-regulated,including NMDA receptor, dopamine D1 receptor, GluR-B, etc.. 13 transcripts were down-regulated , such as dopamine D3 receptor, glutamate receptor subunit 4c, and vasopressin V1b receptor, etc. In the signal transduction system, 13 transcripts were up-regulated, and 7 transcripts were down-regulated. Transcripts of potassium channel, sodium channel and calcium channel also showed altered expression. For example, outward rectifer K+channel and Ca2+-activated K+ channel were down-regulated, other voltage-dependent K+ channel including Kv2.3r were up-regulated; B2 subunit of voltage-dependent Na+ channel was increased, whereas a subunit and B1 subunit were decreased; B3 subunit of Ca2+ channel was up-regulated.These results suggested that desensitized nicotinic receptors induced by chronically repeated nicotine treatment could modulate the genes expression of important target protein in brain, including brain membrane receptors, signal transduction systems and ion channels, which may be responsible for the molecular mechanisms involved in nicotine tolerance and addiction.2. Modulation by nAChRs desensitization on activities of cAMP dependent protein kinase (PKA) and protein kinase C (PKC) in rat brainTreated with nicotine, nicotinic receptors were in activated state, sub-acute desensitized state, acute desensitized state or chronic desensitized state respectively. PKA and PKC samples were abstained from the rat whole brains, their activities were assayed by transferring phosphorus (32P) into the biotinylated peptide Neurogranin and Kemptide, the most specific substrate for PKC and PKA. In addition, using GeneChip, we analyzed the changes in gene expression of PKC and PKA related transcripts after chronic treatment with nicotine. The results showed: there were no differences in activities ofPKA and PKC between control rats and nicotine-treated rats when nicotinic receptors were activated, but PKA and PKC activities were decreased when nicotinic receptors were in sub-actue, actue or chronic desensitized states. The data from GeneChips showed: after chronic treated with nicotine, 9 transcripts including a, r, , subunits of PKC, PKC I, cAMP phosphodiesterase , PKA II regulator , PKA inhibitor, CREB, and CREM did not change, whereas PKC III were up-regulated.These results indicated that desensitized nic...
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