No abstract

No abstract

In the course of our study concerning gastrin and cholecystokinin (CCK) receptors, we have synthesized and characterized a new labeled gastrin ligand, 125I‐BH‐[Leu15]‐gastrin‐(5‐17) [(3‐[125I]iodo‐4‐hydroxyphenyl)‐propionyl‐[Leu15]‐gastrin‐(5‐17)]. Binding of 125I‐BH‐[Leu15]‐gastrin‐(5‐17) to isolated canine fundic mucosal cells was specific, saturable and of high affinity. 125I‐BH‐[Leu15]‐gastrin‐(5‐17) and 125I‐BH‐CCK‐8 [(3‐[125I]iodo‐4‐hydroxyphenyl)‐propionyl‐CCK‐8] interact with isolated canine fundic mucosal cells with small differences in maximal binding capacities and affinities, 3800 ± 900 binding sites/cell (Kd = 0.52 ± 0.23 nM) and 6200 ± 1100 binding sites/cell (Kd= 0.31 ± 0.18 nM), respectively. The relative order of potencies for gastrin and CCK analogs in displacing 125I‐BH‐[Leu15]‐gastrin‐(5‐17) binding correlated well with those obtained using 125I‐BH‐CCK‐8. Selective CCK/gastrin antagonists L‐364,718 (MK‐329) and L‐365,260 also inhibited 125I‐BH‐[Leu15]‐gastrin‐(5‐17) binding. These results indicate that 125I‐BH‐[Leu15]‐gastrin‐(5‐17) binds to gastrin receptors in isolated canine fundic mucosal cells. We have also characterized 125I‐BH‐[Leu15]‐gastrin‐(5‐17) binding to the human Jurkat lymphoblastic cell line (Jurkat cells) known to express the CCK‐B/gastrin receptor. Saturation experiments have shown that both 125I‐BH‐[Leu15]‐gastrin‐(5‐17) and 125I‐BH‐CCK‐8 interact with a single class of high‐affinity binding sites in the Jurkat cell line. Binding characteristics of 125I‐BH‐[Leu15]‐Gastrin‐(5‐17) and 125I‐BH‐CCK‐8 were estimated to be about 2500 ± 400 binding sites/cell (Kd= 0.19 ± 0.09 nM) and 2400 ± 350 binding sites/cell (Kd= 0.06 ± 0.02 nM), respectively. Furthermore, the apparent affinities of CCK analogs and selective antagonists MK‐329 and L‐365,260 for the sites labeled by both probes were identical. The biological activity of cold 125I‐BH‐[Leu15]‐gastrin‐(5‐17) and [Leu15]‐gastrin‐(5‐17) was demonstrated by their ability to increase intracellular calcium concentration in Jurkat cells. All these experiments showed that 125I‐BH‐[Leu15]‐gastrin‐(5‐17) provides a convenient ligand for gastrin receptor binding studies. © Munksgaard 1994.

Germain est ingénieur, chef de projet à l'École de l'ADN Véronique Beaumes est rédactrice en chef au musée d'Histoire naturelle de Nîmes Jean-Christophe Lallement est docteur en Biochimie, Biologie cellulaire et moléculaire, directeur de École de l'ADN Frédéric Delsuc est chargé de recherches (CR1) au

Cholecystokinin (CCK) is one of the most widely distributed brain neuropeptides. It occurs in the whole nervous system in several molecular forms in which CCK-8 predominates.' Cholecystokinin and gastrin share the same C-terminal pentapeptide sequence, and proper gastrin-17 immunoreactivity have also been found in the central nervous system and medulla oblongata in small but significant amounts.24 Cholecystokinin binding to brain tissue preparations was the subject of several studies, leading to good knowledge of CCK receptor repartition and pharmacology throughout the nervous s y~t e m .~ Binding experiments were performed using 1251-CC&,6 [3H]-CC&: 12SI-CCK33,8 as well as [3H]-CC&9 and [3H]pentagastrinlo which represent the common C-terminal sequence of CCK and gastrin and are selective CCKB radioligands. Binding studies using longer forms of gastrin were performed with various gastrin ligands in which the tyrosine residue located in the C-terminal part of the molecule was labeled by IEI. Iodination of tyrosine-12 in Gastrine-17 or gastrin analogs leads to labeled ligands that might interfere with specific properties of gastrin compared to CCK, because the position of the tyrosine is crucial in gastrin and CCK molecules. Therefore, the use of 12SI-BH-[Leu'5]-gastrin-(5-17), a newly synthesized probe having the biological activity of gastrin and in which the integrity of tyrosine located in the C-terminal part of the molecule have been respected," led us to the characterization of specific gastrin-binding sites on guinea pig brain membranes. Recent works after the cloning purification of CCKB receptor (CCKBR) and gastrin receptor (GR) from various mammal t i s~u e s '~J~ strongly suggested that CCK-B and gastrin receptors are the same receptors.Indeed, 1251-BH-[Leu15]-gastrin-(5-17) binds, on guinea pig brain membranes, to a homogeneous population of binding sites (Bmm is about 17.3 * 3.3 fmol/mg of protein) with high affinity (K,, = 0.53 2 0.03 nM) (FIG. 1). The nonpeptide antagonists L-365,260, selective for CCKBR, and MK-329, selective for CCKAR, inhibited 'ZSI-BH-[Leu15]-gastrin-(5-17) binding with ICs,,' s of 6.5 2 0.9 nM and 96 2 15 nM, respectively, in accordance with their selectivity. '2SI-BH-[Leu15]-gastrin-(5-17) binding is completely inhibited by CCK-8 as well as the unlabeled homologous peptide.[Leu15]-gastrin-(5-17) is able to completely inhibit lZI-BH-CC& binding. These inhibition-competition studies showed that 1EI-BH-[Leu15]-gastrin-(5-17) bound to the CCKBR. However, '2SI-BH-[Leu1S]-gastrin-(5-17) and l3I-BH-CC& binding were compared, and Scatchard studies indicated that lZI-BH-CC& binds to a single class of high-affinity binding sites (Kd = 0.045 ? 0.007 nM; B, , is about 50.6 2 8.4 fmol/mg of protein) (FIG. 1). lZI-BH-CC& and 12SI-BH-[Leu15]-gastrin-(5-17) bind 346