Despite the plethora of data indicating beneficial effects of angiotensin (1-7) (Ang 1-7) on the cardiovascular system, its putative receptor, Mas, has not been characterized in tissue membrane preparations other than single concentration demonstrations of the localization of
125
I-Ang 1-7 binding sites in rat kidney. This does not indicate the specificity of
125
I-Ang 1-7 binding nor does it indicate the actual densities of the binding sites, i.e., B
max
(fmoles/mg tissue), or dissociation constant (K
D
) to indicate binding affinity of
125
I-Ang 1-7 for its putative receptor. To characterize
125
I-Ang 1-7 binding in the kidney we prepared a low specific activity, monoradioiodinated Ang 1-7 using a 1:19 mix of
125
iodine :
127
iodine which allows for assessment of the B
max
and K
D
with concentrations of radioligand up to 100 nM. Frozen kidneys from adult male albino rats were dissected and homogenized in water and the membranes were precipitated by centrifugation at 48 kxG. Membranes were resuspended in Tris:MgCl
2
(50:1) pH 7.2 and incubated with 12 concentrations of
125/127
I-Ang 1-7 ranging from ~3-100 nM for 30 min at 22 C, after which bound
125/127
I-Ang 1-7 was resolved from unbound
125/127
I-Ang 1-7 by filtration and measured with a gamma counter. Specific binding (defined as 100 μM Ang 1-7 displaceable binding) of
125/127
I-Ang 1-7 showed a moderate binding affinity (K
D
= 14.7 ± 1.8 nM) and binding site density (B
max
= 24.5 ± 9.9 fmoles/mg initial wet weight). The B
max
value tended to be lower than that in the liver (B
max
= 62.3 ± 20.1 fmoles/mg initial wet weight) and the K
D
value was significantly greater (lower affinity) than that in the liver ( K
D
= 5.7 ± 0.6 nM, p = 0.0085). Of note, competition for
125/127
I-Ang 1-7 binding Ang 1-7 indicated that the IC
50
for Ang 1-7 competition for
125/127
I-Ang 1-7 binding was 42.5 μM. Moreover, the ability of a variety of angiotensin peptides to inhibit
125/127
I-Ang 1-7 binding at 100 μM, Ang 1-7 was less potent that the other angiotensin peptides: Ang III > Ang II > Ang I ~ Ang IV > Ang 2-7 > Ang 1-7 ~ Ang 3-7. These studies suggest that the binding site for
125/127
I-Ang 1-7 is not specific for the putative Ang 1-7 receptor mas, and may represent a low affinity binding to the AT
1
or AT
2
receptor
Identification of a second putative receptor of platelet-activating factor from human polymorphonuclear leukocytes.
