A method for the preparation of mononuclear cells devoid of platelet contamination and its application to the evaluation of putative alpha-receptors in normal and asthmatic subjects.
Receptor studies of human mononuclear leukocytes (MNLs) are complicated by the presence of contaminating platelets which have common receptors. A method was devised to produce MNLs free of platelets (less than 1%) and consists of sequential Ficoll-Hypaque gradients, a BSA gradient and a washing step. Lack of platelet contamination was confirmed by the following criteria: (a) microscopic evaluation using fluorescent dyes showed less than 1% platelets; (b) PGE1 stimulation of the leukocyte membrane adenylate cyclase required addition of exogenous GTP while the platelet cyclase did not; (c) immunoblots of the cells and membranes using antibodies strongly reactive against platelet membranes showed no reactivity against MNL membranes; (d) [3H]yohimbine showed no binding in MNL membranes under conditions where substantial binding to platelets was detected. MNLs were viable as judged by dye exclusion. PHA stimulation of lymphocytes was unimpaired. Plasma membranes of MNLs were prepared by brief sonication and fractionation on a sucrose step gradient. Binding studies using 3H-DHE, an alpha-receptor ligand, revealed no binding in MNLs from normal subjects (n = 6). By contrast, studies on cells from subjects with mild asthma with medication appropriately withheld (n = 8) showed low levels of binding (60-300 fmol/10(6) cells). The subtype and functionality of the putative alpha-receptors are being further evaluated.
Lad
P M
PM
Kaiser Permanente Regional Research Laboratory, Los Angeles, CA 90027.
Easton
J
J
Niedzin
H
H
Olson
C V
CV
Goldberg
B J
BJ
Kaplan
M
M
Parkman
R
R
Learn
D B
DB
Reisinger
D M
DM
D000262:Adenylyl Cyclases / Q000097:blood; D000328:Adult; D001249:Asthma / Q000097:blood*; D001792:Blood Platelets / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism*; D011942:Receptors, Adrenergic, alpha / Q000032:analysis*; D012710:Serum Albumin, Bovine; D015016:Yohimbine / Q000097:blood
D011942:Receptors, Adrenergic, alpha; D012710:Serum Albumin, Bovine; D015016:Yohimbine; D000262:Adenylyl Cyclases