Photoaffinity labeling with dihydropyridine derivatives of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain.
The characteristics of photoaffinity labeling with the calcium agonist [3H]Bay K 8644 (Bay) and the calcium antagonists [3H]nitrendipine (Nit) and (+)PN200-110 (PN) of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain were investigated. In all these crude membranes, [3H](+)PN (20 nM) was mainly photoincorporated into one protein band with a molecular weight of 30,000 - 41,000 Da. It was also incorporated into some other bands of all these crude membranes. The photoincorporation of [3H](+)PN into these crude membranes was inhibited by the presence of 20 microM unlabeled (+)PN. The photoincorporation of [3H](+)PN into these crude membranes depended on its dose and on the time of UV irradiation. No incorporation of [3H](+)PN was observed in the absence of UV irradiation. The incorporation was not affected by the presence of 1 mM CaCl2 and/or 0.15 M NaCl, but was significantly decreased by 20 microM (+)PN and slightly decreased by 20 microM (-)PN, 20 microM Bay, 1 mM diltiazem, or 1 mM verapamil. Namely, enantiomers of PN caused various extents of stereoselective inhibition of photoaffinity labeling by [3H](+)PN of specific protein bands in these crude membranes. [3H]Nit was photoincorporated into these crude membranes in the same way as [3H](+)PN, but [3H]Bay was not photoincorporated. However, 20 microM unlabeled Nit did not consistently inhibit photoaffinity labeling with [3H]Nit. These findings suggested that measurement of photoaffinity of crude membranes from rat skeletal, cardiac, and uterine muscles and whole brain with [3H](+)PN by UV irradiation is a useful method for investigating the characteristics of the voltage-dependent calcium channels that are affected by 1,4-dihydropyridine derivatives.
Ichida
S
S
Department of Biological Chemistry, Faculty of Pharmacy, Kinki University, Osaka.
Masada
A
A
Fujisue
T
T
Yoshioka
T
T
Matsuda
N
N
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000378:metabolism*; D004095:Dihydropyridines / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007082:Ileum / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008566:Membranes / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism; D009132:Muscles / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D009568:Nitrendipine / Q000378:metabolism; D051381:Rats; D013050:Spectrometry, Fluorescence; D014599:Uterus / Q000378:metabolism
D000345:Affinity Labels; D004095:Dihydropyridines; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D009568:Nitrendipine