Kinetics of alkylation of cloned rat alpha1-adrenoceptor subtypes by chloroethylclonidine.

L Xiao, W B Jeffries

Index: Eur. J. Pharmacol. 347 , 319-327, (1998)

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Abstract

We quantified and compared the rates at which chloroethylclonidine (CEC) inactivated cloned rat alpha1A, alpha1B-, and alpha1D-adrenoceptors. Membranes from cells transfected with one of the three cloned alpha1-adrenoceptors were incubated for various intervals with 100 microM chloroethylclonidine at 10 degrees C, 25 degrees C or 37 degrees C. The fraction of receptors alkylated by chloroethylclonidine was determined by [3H]prazosin binding. Chloroethylclonidine fully inactivated each alpha1-adrenoceptor subtype via a first order reaction. Alkylation by chloroethylclonidine was markedly slower for the alpha1A-adrenoceptor vs. the other two subtypes (rate constants in 10(-3) min(-1) at 10 degrees C: 0.99 +/- 0.01 (alpha1A), 7.26 +/- 0.15 (alpha1B), and 7.01 +/- 0.12 (alpha1D)). Despite differences in rate, activation energies for alkylation were similar among subtypes. suggesting a similar binding sites for chloroethylclonidine. Computer simulations of kinetic data in mixed receptor populations and experiments with membranes from rat brain showed that nonlinear curve fitting could distinguish relative proportions of alpha1A-adrenoceptor vs. the other two subtypes. We conclude that measurement of the rate of alkylation by chloroethylclonidine, rather than the total amount of alkylation, is most useful in distinguishing the relative proportion of alpha1A-adrenoceptor in tissues.