Abstract
Aims: In engineered cells, endothelin ETA and ETB receptors can heterodimerize. We tested whether this can also be observed in native tissue. Main methods: Rat mesenteric resistance arteries (rMRA) were maintained in organ culture for 24 h to upregulate ETB-mediated contractions in addition to their normal ETA-mediated responses. They were then exposed to 100 nM linear ET-1 (ETB-agonist) labeled with Oregon Green 488 (OG488/L.-ET-1) and/or to 16 nM intact ET-1 (ETA/ETB-agonist) labeled with the rhodamine dye TAMRA (TAMRA/ET-1). Two photon laser scanning microscopy (TPLSM) was used for the visualization of their binding in the tissue. Fluorescence Lifetime Imaging Microscopy (FLIM) was employed for measurements of the OG488/L-ET-1 lifetime in the absence and presence of TAMRA/ET-1. Key findings: After incubation with the labeled ligands, medial smooth muscle cells (SMCs) were efficiently stained and became visible under TPLSM. TAMRA/ET-1 bound to all SMCs whereas OG488/L-ET-1 stained only groups of SMCs. Interaction of the two receptor subtypes in SMC was investigated in double staining experiments. Fluorescence lifetime of OG488/L.-ET-1 was reduced in the presence of TAMRA/ET-1, which indicates the occurrence of Fluorescence Resonant Energy Transfer (FRET) and suggests close proximity of the two receptor subtypes within the arterial wall. Significance: The methodology that is introduced by these new observations may be useful to assess ET-receptor heterodimerization in biopsies from relevant experimental animal models and human patients.
Original language | English |
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Pages (from-to) | 36-41 |
Journal | Life Sciences |
Volume | 111 |
Issue number | 1-2 |
DOIs | |
Publication status | Published - 28 Aug 2014 |
Keywords
- Endothelin
- Endothelin receptors
- Resistance arteries
- FLIM
- FRET
- Two photon microscopy