The effects of sulfhydryl-modifying reagents on cholecystokinin-receptor interactions in guinea pig gastric glands.

Abstract

The effects of a number of different sulfhydryl agents on cholecystokinin (CCK)-binding sites in isolated fundic gastric glands and gastric mucosal membranes from guinea pigs were evaluated. [125I]CCK octapeptide binding was significantly reduced when gastric glands were pretreated with iodoacetamide (IA; 10 mM), p-chloromercuribenzoate (PCMB; 0.1 mM), or N-ethylmaleimide (NEM; 0.1 mM) at 24 C, but not by dithiothreitol (DTT; 1.0 mM) or dinitrothiobenzoic acid (DTNB; 10 mM). In contrast, inclusion of DTT or IA during binding led to significant increases in the amount of radioligand bound, while in the presence of DTNB binding was significantly reduced. Glycine-HCl wash of gastric glands pretreated with these agents indicated that NEM and IA reduced radioligand internalization by about 80% (P less than 0.001) and increased surface binding by 20% (P less than 0.01) and 76% (P less than 0.001), respectively. PCMB inhibited radioligand binding to surface sites by more than 90% and completely inhibited radioligand internalization. Gastric mucosal membranes bound significantly less radioligand after PCMB (80%) and NEM (26%) treatment compared to controls, and significantly more after IA treatment (162%). Scatchard analysis of the CCK-receptor interaction indicated two binding sites with dissociation constants of 0.71 and 11.7 nM, and binding capacities of 3.6 and 15.8 nmol/mg DNA, respectively. NEM and IA significantly increased the Kd of the high affinity site without affecting the binding parameters of the low affinity site; the low affinity site was eliminated by PCMB treatment, and the affinity and capacity of the high affinity site were significantly reduced. In the presence of DTT, [125I]CCK octapeptide binding to gastric glands was enhanced with a significant increase in the amount of radioligand in the internalized pool (39%; P less than 0.001) and no increase in binding to gastric mucosal membranes. These results suggest that sulfhydryl groups play an important role in CCK-receptor interactions.