Improved method for autoradiographic localization of beta-adrenoceptors using photoaffinity labelling.
Contact autoradiography of tissue sections, using emulsion coated coverslips or X-ray films, is widely used to provide information about the regional distribution of receptors. This easy to perform, standard technique has the disadvantage of an image spread due to the gap between the radioactive source and the film. The present study describes a new technique which combines photoaffinity labeling of beta-adrenoceptors with "dipping" autoradiography and a modified trichrome stain. Incubation of 16 microns cryosections of rat lung tissue with the iodinated, photoaffinity labeling, non-selective, beta-adrenergic agonist [125I]-cyanopindololazide II ([125I]-CYPA II) (100 pmol/l) in the absence or presence of 1 mumol (+/-)-propranolol revealed strong, specific beta-adrenoceptor binding to alveolar parenchyma and bronchial epithelium of large and small bronchioles, lesser binding to smooth muscle bundles of large airways and only sparse binding to the smooth muscle of small bronchioles or peripheral branches of pulmonary artery. With standard autoradiographic techniques, a similar distribution of the label was obtained, although resolution and sensitivity were inferior. Staining of tissue sections through the photoemulsion by means of a modified Mallory's trichrome dye facilitated the discrimination between alveolar and bronchial epithelium, muscular and collagenous tissues. In conclusion, the photoaffinity labeling of beta-adrenoceptors with [125I]-CYPA II allows the use of "dipping" autoradiography. This technique, in combination with trichrome staining through the photoemulsion, results in an improved autoradiographic image together with a better association of the label with distinct histological structures and the higher sensitivity of the method.
Institut für Toxikologie, Medizinische Hochschule Hannover, Germany.
L
L
Hesse
G
G
Erdmann
T
T
Eschenhagen
H H
HH
Wellhöner
D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography / Q000379:methods*; D001386:Azides; D001391:Azo Compounds; D004396:Coloring Agents; D016616:Cryoultramicrotomy; D004801:Eosine Yellowish-(YS); D004848:Epithelium / Q000378:metabolism; D008168:Lung / Q000378:metabolism*; D008297:Male; D008739:Methyl Green; D009130:Muscle, Smooth / Q000378:metabolism; D010781:Photography; D010869:Pindolol / Q000031:analogs & derivatives; D051381:Rats; D017207:Rats, Sprague-Dawley; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D001391:Azo Compounds; D004396:Coloring Agents; D011943:Receptors, Adrenergic, beta; C061987:cyanopindolol azide-2; C017768:trichrome stain; D008739:Methyl Green; D010869:Pindolol; D004801:Eosine Yellowish-(YS)