guanylyl-imidodiphosphate and Colonic-Neoplasms

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.

Topics: Adenocarcinoma; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Cell Line; Cell Membrane; Clonidine; Colonic Neoplasms; Epinephrine; Guanylyl Imidodiphosphate; Humans; Receptors, Adrenergic, alpha; Yohimbine

Sodium and GppNHp, a GTP analogue, decrease the specific binding of the alpha 2-adrenergic agonist [3H]clonidine on membranes from the HT29 cells; affinity of the ligand for the receptor is also decreased. The effects of the two agents are additive. By contrast, combination of sodium and GppNHp increases the number of binding sites for the alpha 2-adrenergic antagonist [3H]yohimbine. Sodium and GppNHp also decrease the potency of epinephrine and clonidine to displace [3H]yohimbine from the alpha 2-adrenoceptors of the HT29 cells

Topics: Adenocarcinoma; Binding, Competitive; Cell Line; Clonidine; Colonic Neoplasms; Epinephrine; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Receptors, Adrenergic, alpha; Sodium; Yohimbine