Characterization of Cholecystokinin Binding Sites in Rat Cerebral Cortex Using a 125I‐CCK‐8 Probe Resistant to Degradation

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Abstract
Specific binding sites for cholecystokinin (CCK) were characterized in a particulate membrane fraction of rat cerebral cortex using a biologically active 125I-labeled derivative of the C-terminal octapeptide of CCK (CCK-8) prepared by reaction with the iodinated form of the imidoester (125IIE), methyl-p-hydroxybenzimidate. The time course of binding to cortical membranes was rapid, temperature dependent and saturable. Half-maximal binding at 24.degree. C was reached in 30 min and full binding at 120 min. At 37.degree. C there was only a slight increase in 125IIE-CCK-8 bound after 15 min. The addition of a large excess of CCK-8 after 30 min of binding at 24.degree. C caused a prompt and rapid decline in radioligand bound showing that the interaction was reversible. There was a progressive decline in the amount of 125IIE-CCK-8 bound to membranes with increasing concentrations of CCK-8 and other structurally related peptides. CCK-8 displaced 50% of the radioligand at 4 nM, CCK-33 at 10 nM and gastrin (desulfated CCK-8) at 60 nM. Secretin, a structurally unrelated peptide, was unable to displace the radioligand from cortical membranes at 1.0 .mu.M. 125IIE-CCK-8 exposed to cortical membranes or to buffers that had previously contained such membranes for 60 min at 24.degree. C bound equally as well to fresh cortical membranes as control radioligand that had not been exposed to the same conditions. The 125I-CCK-8 radioligand used was highly resistant to degradative processes in rat brain tissue.