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

Dimitrios Kapsokalyvas, Paul M H Schiffers, Nathan Maij, Dennis P Suylen, Tilman M Hackeng, Marc A M J van Zandvoort, Jo G R De Mey

Research output: Contribution to journalJournal articleResearchpeer-review


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 100 nM 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.

Original languageEnglish
JournalLife Sciences
Issue number1-2
Pages (from-to)36-41
Publication statusPublished - 28. Aug 2014


  • Animals
  • Carboxylic Acids
  • Endothelin-1
  • Male
  • Mesenteric Arteries
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Organ Culture Techniques
  • Protein Multimerization
  • Rats
  • Rats, Inbred WKY
  • Receptor, Endothelin A
  • Receptor, Endothelin B
  • Rhodamines
  • Endothelin
  • Two photon microscopy
  • FLIM
  • Endothelin receptors
  • Resistance arteries
  • FRET


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