Abstract
In contrast to reversible activation by agonist, cholecystokinin 1 receptor (CCK1R) is permanently activated by singlet oxygen generated in photodynamic action, with sulphonated aluminium phthalocyanine or genetically encoded mini singlet oxygen generator (miniSOG) as photosensitizer. In these works, a halogen light source was used to power photodynamic action. For possible in vivo application of photodynamic CCK1R physiology, bearing a cumbersome light-delivery device connected to an external light source by experimental animals might interfere with their behavior. Therefore, in the present work, the possibility of bioluminescence-driven miniSOG photodynamic CCK1R activation was examined, as monitored by Fura-2 calcium imaging. In parallel experiments, it was found that, after plasma membrane (PM)-localized expression of miniSOGPM in AR4-2J cells, light irradiation with blue light-emitting diode (LED) (450 nm, 85 mW·cm−2, 1.5 min) induced persistent calcium oscillations that were blocked by CCK1R antagonist devazepide 2 nM. NanoLuc was expressed bicistronically with miniSOGPM via an internal ribosome entry site (IRES) sequence (pminiSOGPM-IRES-NanoLuc). The resultant miniSOGPM-IRES-NanoLuc-AR4-2J cells were found to generate strong bioluminescence upon addition of NanoLuc substrate coelenterazine. Strikingly, coelenterazine 5 microM was found to trigger long-lasting calcium oscillations (a hallmark for permanent CCK1R activation) in perifused miniSOGPM-IRES-NanoLuc-AR4-2J cells. These data indicate that NanoLuc bioluminescence can drive miniSOGPM photodynamic CCK1R activation, laying the foundation for its future in vivo applications.
Highlights
The rhodopsin-type or A class calcium-mobilizing G protein-coupled receptor (GPCR) cholecystokinin type 1 (CCK1 or cholecystokinin 1 receptor (CCK1R)) [1,2] is widely expressed both in the central nervous system (CNS) [3,4,5,6] and in peripheral tissues [7,8,9,10,11,12]
We have previously found that photodynamically generated 1O2 with chemical photosensitizers sulphonated aluminum phthalocyanine (SALPC) or gadolinum porphyrin-like macrocycle B (GdPLMB) permanently activated CCK1R endogenously expressed in rat pancreatic acinar cells [29,30,31] or ectopically expressed in human embryonic kidney epithelial cell HEK293 [33]
We have found that the genetically-encoded protein photosensitizers (GEPPs) KillerRed or mini singlet oxygen generator (miniSOG) expressed at the plasma membrane in rat pancreatic acinar tumor cell AR4-2J were able after light irradiation with a halogen cold light source to photodynamically activate CCK1R permanently [33]
Summary
The rhodopsin-type or A class calcium-mobilizing G protein-coupled receptor (GPCR) cholecystokinin type 1 (CCK1 or CCK1R) [1,2] is widely expressed both in the central nervous system (CNS) [3,4,5,6] and in peripheral tissues [7,8,9,10,11,12]. Emergence in recent years of genetically-encoded protein photosensitizers (GEPPs) such as KillerRed or mini singlet oxygen generator (miniSOG) prompted us to extend our earlier works with chemical photosensitizers to these new GEPPs. We have found that the GEPPs KillerRed or miniSOG expressed at the plasma membrane in rat pancreatic acinar tumor cell AR4-2J were able after light irradiation with a halogen cold light source to photodynamically activate CCK1R permanently [33]
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