Cellular distribution of endothelin-1 mRNA in human brain by in situ RT-PCR.
Endothelins (ETs) are a family of potent vasoconstrictor and comitogenic polypeptides consisting of 21-amino acids. Using in situ hybridization, ET-1 mRNA has previously been localized to neuronal cell bodies in fourteen human brain regions. However, because in situ hybridization has a limited detection sensitivity of 20 mRNA copies per cell, ET-1 mRNA may be present in previously undetected areas. Hence, our objective was to localize ET-1 mRNA in specific human brain regions and astrocytic tumours using the more sensitive in situ reverse transcriptase polymerase chain reaction (in situ RT-PCR). Human brain autopsy tissue and surgical cerebral tumour tissue were treated with proteinase K and DNase, followed by RT-PCR using primers specific for ET-1 mRNA and digoxygenin-labelled dUTP in the PCR mixture. The DIG-dUTP was localized with an immunodetection system. We demonstrate ET-1 mRNA labelling in twenty two of the twenty four brain regions studied including those regions in which ET-1 mRNA has been observed by in situ hybridization. In addition, the localization of ET-1 mRNA observed in astrocytomas suggests a role for ET-1 in tumour pathogenesis. In situ RT-PCR has proven to be highly sensitive in its ability to detect low mRNA expression at the cellular level. Our results confirm a role for ET-1 in the human nervous system.
Naidoo
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V
Department of Pharmacology, Nelson R Mandela School of Medicine, University of Natal, Durban, Congella, South Africa.
Mahabeer
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R
Raidoo
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DM
D001254:Astrocytoma / Q000737:chemistry* / Q000209:etiology; D001923:Brain Chemistry; D001932:Brain Neoplasms / Q000737:chemistry; D019332:Endothelin-1 / Q000032:analysis* / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D019332:Endothelin-1; D012333:RNA, Messenger