Development of Region-specific Radioimmunoassays against Rat Prosecretin or Secretin and their Characterization

Abstract

We have raised four region-specific antisera in rabbits against rat prosecretin or secretin using synthetic peptides such as prosecretin(]--41), prosecretin(Z-]]), prosecretin(98~]13) and secretin and developed three region-specific radioimmunoassays (RIAs) for prosecretin(]-41), prosecrtin(1-11) and secretin. The results showed clearly the presence of prosecretin-like peptides in rat gastrointestine, whereas only a trace of prosecretin-like IR were recognized, if any, in the brain. In the Wistar male rat gastrointestine, the highest concentration of both prosecretin(1~41)-like and secretin-like immunoreactivity (IR) were detected in the ileum rather than in the duodenum. It is noteworthy that serial section of rat deodenum revealed the positive staining of prosecretin(98--113)-like IR in the cells identical to those stained with both anti-prosecretin(l-4]) serum and anti-secretin serum. These result, clearly indicate the presence of the C-terminal fragments of prosecretin as the processing products. In contrast, anti-prosecretin(]-1]) serum did not recognize the immunoreactivity in any ofthe cells in the brain and gastrointestine.

Expression of Secretin in Porcine and Rat Central Nervous System

“…The content in the other regions was very low, nevertheless antibodies RY28 and R0311 recognized secretin in a similar manner. Our previous report 19) failed to detect secretin-LI in the rat brain with antibody RY28, as the quantity of tissue used might have been too small. This lead to the speculation that low production and restricted localization of secretin in the CNS caused apparent inconsistent conclusions be- tween investigators.…”

“…This lead to the speculation that low production and restricted localization of secretin in the CNS caused apparent inconsistent conclusions be- tween investigators. 6,[16][17][18][19][20] In this paper, secretin-LI was extracted and concentrated from a large amount of porcine cortex to investigate the proforms of secretin and to confirm the production of secretin in the CNS. Second, secretin-positive cells were thoroughly searched for in the rat brain histochemically.…”

“…Antibody RY28 was raised against synthetic rat secretin or prosecretin (12-38) amide and was specific for the C-terminal amide region, showing no cross-reactivity with Cterminally extended forms. 19) Antibody R0311 was raised against synthetic rat prosecretin (1-41) and equally recognized the C-terminal amide form and the C-terminally extended form of secretin. 19) Gel filtration chromatography and HPLC of porcine cortex extracts revealed at least four kinds of proforms in addition to secretin.…”

“…Radioimmunoassay ---Rabbit antibodies, RY28 against rat secretin or prosecretin (12-38) amide, and R0311 against rat prosecrein (1-41), were used for radioimmunoassay (RIA). 19) Rat secretin and antibody RY28 were generously provided by Dr. N. Yanaihara. Antibody R0311 recognized both secretin and N-terminally extended proforms on an equimolar basis.…”

“…gion of the CNS by an antibody against secretin. 19) In terms of secretin cDNA, Northern blot hybridization with high stringency showed only trace levels of secretin mRNA in the rat hypothalamus, brainstem and cortex, although secretin-LI was detected in several regions of the CNS. 16) In contrast, polymerase chain reaction showed a wide distribution of secretin mRNA in the rat CNS, 6,20) whereas secretin-LI could not be detected in the rat brain.…”