m1-m5 muscarinic receptor distribution in rat CNS by RT-PCR and HPLC
| Autor: | Elizabeth A. Walton, Jerry J. Buccafusco, Jian Wei, Antonio Milici |
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| Rok vydání: | 1994 |
| Předmět: |
Male
Molecular Sequence Data Biology Glucosephosphate Dehydrogenase Biochemistry Polymerase Chain Reaction Cellular and Molecular Neuroscience chemistry.chemical_compound Complementary DNA Muscarinic acetylcholine receptor Animals Tissue Distribution RNA Messenger Rats Wistar Chromatography High Pressure Liquid Gel electrophoresis Brain Chemistry Messenger RNA Base Sequence RNA-Directed DNA Polymerase Molecular biology Receptors Muscarinic Reverse transcriptase Rats genomic DNA Real-time polymerase chain reaction chemistry Spinal Cord Ethidium bromide |
| Zdroj: | Journal of neurochemistry. 63(3) |
| ISSN: | 0022-3042 |
| Popis: | Five muscarinic receptor genes (m1-m5) that encode distinct muscarinic receptor subtypes have been cloned. Because of their structural homology and pharmacological similarity, ligand binding probes currently available do not clearly distinguish among the subtypes. To obtain a clear distribution within the CNS of molecularly defined muscarinic receptor subtypes, seven brain regions were examined for the expression of the respective mRNAs. The most sensitive method for detecting mRNA is through amplification of the respective cDNAs. Brain regions were obtained from male Wistar rats, and total RNA was isolated. The isolates were extensively treated with RNase-free DNase to remove any residual genomic DNA. Total RNA (1 microgram) was reverse-transcribed using random primers and reverse transcriptase. The resulting cDNA was amplified using a thermal cycler, and the polymerase chain reaction (PCR)-amplified products were analyzed by gel electrophoresis containing ethidium bromide and visualized with fluorescent illumination. PCR-amplified samples were also injected directly onto an HPLC anion exchange column and quantified by UV detection. Each of the five muscarinic subtypes was found in every brain region examined. The m1 subtype was most abundant in cortex and gradually declined in content caudally to the spinal cord. The m2 subtype was most abundant in thalamus-hypothalamus and ponsmedulla. The m4 subtype was found in greatest amount in the striatum, whereas m3 and m5 were expressed consistently throughout the CNS. The combination of RT-PCR and HPLC provides a rapid and sensitive method for quantifying the expression of mRNA coding for all five muscarinic receptor subtypes derived from the CNS. |
| Databáze: | OpenAIRE |
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