Characterization of two different affinity B2-kinin binding sites in rat glomeruli.
We have recently characterized a bradykinin (BK) receptor in rat renal mesangial cells (1). Activation of this receptor is associated with PGE2 release and IP3 formation suggesting involvement in cell contraction which can be linked to the control of the glomerular filtration rate (2). Whether this mesangial BK receptor is the unique glomerular BK receptor remains to be elucidated. In an attempt to answer to this question, we performed binding studies using decapsulated isolated glomeruli. Scatchard analysis of the binding data obtained with this preparation revealed the presence of two distinct B2-kinin binding sites. However, a consistent difference was observed in both the affinity and the density. We further investigated the pharmacological binding profile after an initial step of solubilization. Several experiments were performed to establish optimal conditions of solubilization. For this, different detergents such as Triton X-100, CHAPS and n-octyl beta-D glucopyranoside were tested at various concentrations, durations and temperatures of incubation. The binding was performed with two different [125I]-Tyr0-BK concentrations (0.5 and 7 nM) with either untreated decapsulated glomeruli or solubilized preparation for 45 minutes at +4 degrees C in the binding buffer containing a mixture of protease inhibitors. The greatest binding was achieved after treating glomeruli with 25 mM n-octyl beta-D glucopyranoside for 60 minutes at +4 degrees C under constant shaking. Two B2-kinin receptors of different affinities were detected. The same binding characteristics were obtained both in the 12,000 x g and 100,000 x g supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)
Emond
C
C
INSERM U133, Institut Louis Bugnard, Faculté de Médecine Rangueil, Toulouse, France.
Pécher
C
C
Bascands
J L
JL
Regoli
D
D
Girolami
J P
JP
D000818:Animals; D001667:Binding, Competitive; D001920:Bradykinin / Q000031:analogs & derivatives / Q000378:metabolism*; D003902:Detergents; D007672:Kidney Cortex / Q000378:metabolism*; D007678:Kidney Glomerulus / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification / Q000378:metabolism*
D003902:Detergents; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; C026201:bradykinin, Tyr-; D001920:Bradykinin