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Functional cholecystokinin receptors are distinguished kinetically by biotinyl-Tyr-Gly-(Thr28,Nle31)CCK(25-33) in rat pancreatic acini

par Winand, Jacques ;Poloczek, Piotr ;Delporte, Christine ;Moroder, Luis;Svoboda, Michal ;Christophe, Jean
Référence Biochimica et biophysica acta, 1080, 2, page (181-190)
Publication Publié, 1991-10

Article révisé par les pairs

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Résumé :

Biotinyl-tyrosine-glycine(Thr28,Nle31)CCK(25-33) (BTG-TN-CCK-9) promoted amylase secretion and phosphatidylinositol (PI) metabolism with the same potency and efficacy as TN-CCK-9 in dispersed rat pancreatic acini. A 1 min preincubation of the ligand with a 20-fold excess of streptavidin completely suppressed this biological activity. On the other hand, amylase secretion and PI metabolism prestimulated with BTG-TN-CCK-9 were blocked within 1-5 min after streptavidin addition. [125I]BTG-TN-CCK-9 bound to high (Kd 0.17 nM) and low (Kd 13 nM) affinity receptors. Its dissociation, in the presence of either streptavidin or TN-CCK-9, showed a rapid component and a slow component. The proportion of tracer dissociating slowly increased with increasing preincubation time as did the proportion of tracer that could not be washed away quickly by acidic treatment, in parallel experiments. This phenomenon occurred less readily at 4 degrees C or in the presence of 1 mM CCCP. In acini preincubated for 30 min with 0.3 nM [125I]BTG-TN-CCK-9 and various concentrations of unlabelled BTG-TN-CCK-9, then washed at neutral pH (in order to eliminate rapidly dissociating ligand preferentially), the tracer displacement curve was shifted leftward, suggesting that rapidly dissociating receptors corresponded to low affinity receptors. When acini were preincubated for 1 min with BTG-TN-CCK-9, then washed at neutral pH with buffer only, we observed residual stimulated secretion over the next 30 min period, that correlated with the BTG-TN-CCK-9 concentration offered during the short preincubation period. This phenomenon was inhibited by streptavidin suggesting that intracellularly accumulated intact BTG-TN-CCK-9 (as shown, by radio-HPLC) promoted residual secretion when free to bind again to cell surface receptors in the absence of streptavidin. Taken collectively, these data suggest the coexistence of at least 2 types (or states) of CCK receptors.