Alpha 2-adrenoceptor in HT29 human colon adenocarcinoma cell-line: study of [3H](-)-adrenaline binding.

The HT29 human adenocarcinoma cell-line was used to investigate the binding of 3H-adrenaline at alpha 2-adrenoceptors. All aspects of the study indicated that alpha 2-adrenoceptors were specifically labeled. 3H-adrenaline bound with high affinity (KD = 2.28 +/- 0.41 nM) to a single population of non-interacting sites. The rank order of adrenoceptor antagonists (yohimbine greater than phentolamine much greater than prazosin) and agonists (UK-14,304 greater than clonidine greater than (-)-adrenaline greater than (-)-noradrenaline greater than (+)-adrenaline greater than (+)-noradrenaline greater than amidephrine) to compete with 3H-adrenaline binding showed that the labeled sites were alpha 2-selective and stereospecific. Comparison of the binding parameters of 3H-adrenaline with those of [3H]clonidine (partial-agonist) and [3H]yohimbine (antagonist) indicated that 3H-adrenaline and [3H]clonidine labeled a similar number of sites (156 +/- 13 versus 175 +/- 21 fmol/mg protein) and that [3H]yohimbine (Bmax = 246 +/- 22 fmol/mg protein) labeled more sites than the 3H-agonists. Data on the inhibition of [3H]yohimbine binding by (-)-adrenaline was better fitted to a two-site model and revealed (1) that the KiL/KiH ratio was higher for (-)-adrenaline than for clonidine (2) that both agonists recognized the same percentage of high-affinity receptors. The results from a kinetic study of 3H-adrenaline binding were apparently inconsistent with the equilibrium data. Both the association and dissociation were bi-exponential, suggesting a relative heterogeneity of the labeled sites. The tritiated physiological full-agonist was moreover able to induce tight-binding. The practical consequences of this property are discussed.