Visualization and tissue distribution of alpha1L-adrenoceptor in human prostate by the fluorescently labeled ligand Alexa-488-silodosin.
OBJECTIVE
Although alpha(1L)-adrenoceptor is recognized as a target of alpha(1) antagonist therapy for benign prostatic hyperplasia, the most common techniques, such as immunohistochemistry and in situ hybridization, are not applicable to examine alpha(1L)-AR vs alpha(1A)-AR tissue distribution because alpha(1L)-AR is now considered another phenotype sharing the alpha(1A)-AR gene and protein molecule. We labeled the alpha(1A) and alpha(1L)-adrenoceptor selective antagonist silodosin (Kissei Pharmaceutical, Matsumoto, Japan) with the fluorophore Alexa Fluor(R) 488 (Alexa-488-silodosin) to visualize alpha(1L)-AR expression.


METHODS
Radioligand binding and functional bioassay experiments were done to assess alpha(1)-AR expression in Chinese hamster ovary cells and human prostate tissues. Confocal imaging was subsequently performed.


RESULTS
Although Alexa-488-silodosin had about 10 times lower affinity for all alpha(1)-AR subtypes than silodosin in binding and functional studies, it had high selectivity to alpha(1A) and alpha(1L)-ARs. Confocal imaging revealed clear localization of fluorescence on the membrane of Chinese hamster ovary cells expressing alpha(1A)-AR but not alpha(1B)-and alpha(1D)-ARs, and in the muscle layer of the human prostate. The fluorescent signal in Chinese hamster ovary cells disappeared in the presence of 3 nM prazosin but fluorescence was observed in the human prostate even in the presence of 100 nM prazosin.


CONCLUSIONS
Alexa-488-silodosin is a powerful fluorescent probe with high selectivity to alpha(1A) and alpha(1L)-ARs. Thus, Alexa-488-silodosin successfully visualizes the site of alpha(1L)-ARs in the muscle layer of the human prostate without losing its distinct pharmacological profile.
Morishima
Shigeru
S
Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, University of Fukui, Eiheiji, Japan.
Suzuki
Fumiko
F
Nishimune
Atsushi
A
Yoshiki
Hatsumi
H
Akino
Hironobu
H
Yokoyama
Osamu
O
Muramatsu
Ikunobu
I
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005453:Fluorescence; D005464:Fluorobenzenes; D007211:Indoles; D008297:Male; D011467:Prostate / Q000737:chemistry* / Q000378:metabolism*; D018340:Receptors, Adrenergic, alpha-1 / Q000032:analysis* / Q000378:metabolism*; D014018:Tissue Distribution
C513915:AlexaFluor 488 carboxylic acid tetrafluorophenyl ester; D005464:Fluorobenzenes; D007211:Indoles; D018340:Receptors, Adrenergic, alpha-1; C095285:silodosin