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Imaging evidence for endothelin ETA/ETB receptor heterodimers in isolated rat mesenteric resistance arteries

Life Sciences
Publication Date
DOI: 10.1016/j.lfs.2014.07.007
  • Endothelin
  • Endothelin Receptors
  • Resistance Arteries
  • Flim
  • Fret
  • Two Photon Microscopy
  • Engineering
  • Physics


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 24h to upregulate ETB-mediated contractions in addition to their normal ETA-mediated responses. They were then exposed to 100nM linear ET-1 (ETB-agonist) labeled with Oregon Green 488 (OG488/L.-ET-1) and/or to 16nM 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.

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