Steven W. Ryder

Immunochemical studies relating to cholecystokinin in brain and gut.

Publisher Summary This chapter presents an overview of the immunochemical studies relating to cholecystokinin in the brain and gut. Cholecystokinin (CCK) is a central nervous system (CNS) peptide. It is exceptional in its high brain concentration—far exceeding the brain concentrations of other brain–gut peptides—and remarkable in its abundance and broad distribution throughout the cerebral cortex. It is further unusual in the variety of its heterogeneous molecular forms; these are distributed differently in brain and gut tissues. Both intact CCK-33 and its COOH-terminal fragments are found in the brain as well as in the gut. The chapter discusses the two discoveries that have contributed to the current surge of interest in immunochemical studies of CCK. The COOH-terminal approach is preferred for the measurement of CCK peptides in gastrin-free tissue extracts but is not applicable to the measurement of plasma CCK because of the relative abundance of gastrin peptides in plasma and their strong cross-reactivity in this assay.

Alkaline extraction and characterization of cholecystokinin-immunoreactivity from rat gut.

Generally some variation of neutral or acid extractants has been used to recover immunoreactive cholecystokinin (CCK) from gut as well as from brain. Recovery of CCK in 0.1 N NaOH extracts from gut mucosa, gut muscle, or whole gut ranged up to threefold higher than in similar water or 0.1 N HCl extracts, although the reverse was the case for the extraction of secretin from the same tissue. CCK-immunoreactive peptides from rat gut were found to resemble a CCK-33-like peptide, sulfated CCK-12 and CCK-8, as well as smaller COOH-terminal fragments, which are larger than the C-terminal tetrapeptide amide. The fraction of immunoreactivity in the form of a CCK-33-like peptide was greater, although the total recovery was less, in acid extractants of whole gut. Proper interpretation of dynamic changes in gut CCK in response to fasting, feeding, and other laboratory manipulations requires efficient extraction of total immunoreactivity.

Alkaline extraction of cholecystokinin-immunoreactivity from rat brain

Abstract Alkaline aqueous extractants remove from rat brain 2 to 4 times the CCK-immunoreactivity that is removed by acidic or neutral aqueous extractants. The distribution among the various hormonal forms appears to be independent of the extractant: about 1 10 in the larger basic forms (CCK-33 and CCK-39); about 1 4 as the C-terminal dodecapeptide (CCK-12) and the remainder as the octapeptide (CCK-8). In contrast, alkaline and acidic aqueous solutions are equally efficient in extraction of enkephalin-immunoreactivity from the same tissues. We are presently unable to account for the very different efficiencies of the various extractants in removing CCK-immunoreactivity from brain.

Cholecystokinin and Enkephalin levels following ethanol administration in rats

Using radioimmunoassay, cholecystokinin and enkephalin levels were determined in the brain and gut of rats following ethanol administration. Acute or chronic administration of ethanol did not affect the cholecystokinin or enkephalin content of rat brain cortex, hypothalamus, striatum or proximal small bowel. This contrasts with the reports of altered levels of several classical neurotransmitters following ethanol administration.

Nature of immunoreactive CCK in rat and pig brain.

Two major classes of immunoreactive cholecystokinin peptides (iCCK) have been identified in rat and pig brains: (1) large basic peptides (Big iCCK) resembling pCCK33 in size and charge; (2) small acidic peptides (Small iCCK) resembling the COOH-terminal fragments of CCK. Boiling 0.1 N HCl maximally extracts Big iCCK; boiling 0.1 N NaOH maximally extracts Small iCCK. The differences in hormonal forms removed by these extractions are not likely to be due to enzymatic conversion during the extraction procedures. Fractionation on Sephadex G50 and starch gel electrophoresis combined with radioimmunoassay using 3 antisera of different specificities: (1) directed towards the NH2-terminus of pCCK33; (2) produced by immunization with CCK8; (3) produced by immunization with CCK4; are consistent with the hypothesis that a major fraction of Big iCCK may represent intact CCK with a COOH-terminus extension as has recently been suggested for gastrin, a molecule having a COOH-terminal pentapeptide identical with that of CCK.