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

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

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.

OriginalsprogEngelsk
TidsskriftLife Sciences
Vol/bind111
Udgave nummer1-2
Sider (fra-til)36-41
ISSN1574-6895
DOI
StatusUdgivet - 28. aug. 2014

Fingeraftryk

Photons
Confocal Microscopy
Fluorescence
Rhodamines
Optical Imaging
Organ Culture Techniques
Microscopy
Coloring Agents
Up-Regulation
Ligands

Citer dette

Kapsokalyvas, D., Schiffers, P. M. H., Maij, N., Suylen, D. P., Hackeng, T. M., van Zandvoort, M. A. M. J., & De Mey, J. G. R. (2014). Imaging evidence for endothelin ETA/ETB receptor heterodimers in isolated rat mesenteric resistance arteries. Life Sciences, 111(1-2), 36-41. https://doi.org/10.1016/j.lfs.2014.07.007
Kapsokalyvas, Dimitrios ; Schiffers, Paul M H ; Maij, Nathan ; Suylen, Dennis P ; Hackeng, Tilman M ; van Zandvoort, Marc A M J ; De Mey, Jo G R. / Imaging evidence for endothelin ETA/ETB receptor heterodimers in isolated rat mesenteric resistance arteries. I: Life Sciences. 2014 ; Bind 111, Nr. 1-2. s. 36-41.
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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 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.",
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author = "Dimitrios Kapsokalyvas and Schiffers, {Paul M H} and Nathan Maij and Suylen, {Dennis P} and Hackeng, {Tilman M} and {van Zandvoort}, {Marc A M J} and {De Mey}, {Jo G R}",
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year = "2014",
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Kapsokalyvas, D, Schiffers, PMH, Maij, N, Suylen, DP, Hackeng, TM, van Zandvoort, MAMJ & De Mey, JGR 2014, 'Imaging evidence for endothelin ETA/ETB receptor heterodimers in isolated rat mesenteric resistance arteries', Life Sciences, bind 111, nr. 1-2, s. 36-41. https://doi.org/10.1016/j.lfs.2014.07.007

Imaging evidence for endothelin ETA/ETB receptor heterodimers in isolated rat mesenteric resistance arteries. / Kapsokalyvas, Dimitrios; Schiffers, Paul M H; Maij, Nathan; Suylen, Dennis P; Hackeng, Tilman M; van Zandvoort, Marc A M J; De Mey, Jo G R.

I: Life Sciences, Bind 111, Nr. 1-2, 28.08.2014, s. 36-41.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Imaging evidence for endothelin ETA/ETB receptor heterodimers in isolated rat mesenteric resistance arteries

AU - Kapsokalyvas, Dimitrios

AU - Schiffers, Paul M H

AU - Maij, Nathan

AU - Suylen, Dennis P

AU - Hackeng, Tilman M

AU - van Zandvoort, Marc A M J

AU - De Mey, Jo G R

N1 - Copyright © 2014 Elsevier Inc. All rights reserved.

PY - 2014/8/28

Y1 - 2014/8/28

N2 - 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.

AB - 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.

KW - Animals

KW - Carboxylic Acids

KW - Endothelin-1

KW - Male

KW - Mesenteric Arteries

KW - Microscopy, Confocal

KW - Microscopy, Fluorescence

KW - Organ Culture Techniques

KW - Protein Multimerization

KW - Rats

KW - Rats, Inbred WKY

KW - Receptor, Endothelin A

KW - Receptor, Endothelin B

KW - Rhodamines

U2 - 10.1016/j.lfs.2014.07.007

DO - 10.1016/j.lfs.2014.07.007

M3 - Journal article

C2 - 25066928

VL - 111

SP - 36

EP - 41

JO - Life Sciences

JF - Life Sciences

SN - 1574-6895

IS - 1-2

ER -

Kapsokalyvas D, Schiffers PMH, Maij N, Suylen DP, Hackeng TM, van Zandvoort MAMJ et al. Imaging evidence for endothelin ETA/ETB receptor heterodimers in isolated rat mesenteric resistance arteries. Life Sciences. 2014 aug 28;111(1-2):36-41. https://doi.org/10.1016/j.lfs.2014.07.007