Abstract
Radioimmunoassay for two hypothalamic peptides, somatostatin (somatotropin-release inhibiting factor: SRIF) and neurotensin (NT), was described. Tyr1-SRIF was radioiodinated by a modification of Chloramine T method and purified on Sephadex G-25 column using acetic acid as an eluant. Labelled tyr1-SRIF was absorbed to Sephadex G-25 and eluted broadly after free 1251. The elution pattern of labelled tyr1-SRIF was dependent to the molar concentration of the eluant. KD value of labelled tyr1-SRIF in each concentration of 1.0M, 0.5M, 0.1M and 0.05M acetic acid was 0.95, 1.61, 2.56 and 3.51, respectively. NT was labelled with 1251 by Chloramine T method. Labelled NT was purified on Sephadex G-25 using phosphate buffer saline with 0.1% gelatin (pH 7.6). In both assays, the separation of bound and free was performed by double antibody method.
Anti-SRIF serum was produced by the method of Arimura et al. and assayed in a final dilution of 1: 4000. The sensitivity of the assay was 10pg/tube. The dilution curves of various rat tissue extracts run parallel with standard curve.
Anti-NT serum was generated by immunizing rabbits with neurotensin-bovine thyroglobulin conjugates according to the method of Carraway and Leeman. In the radioimmunoassay, anti-NT serum was finally diluted at 1: 56000. The minimum detectable level was 10pg/tube. The dilu- tion curves of different regional extracts of the rat brain gave excellent parallerism with standard curve.
Brain and gastrointestinal distribution of immunoreactive SRIF (IR-SRIF) and NT (IR-NT) was examined in adult male rats. Tissues were extracted with 1ml of acetone per 100mg tissue weight. Total contents of brain IR-SRIF and IR-NT were 89.8±3.2ng and 71.6±1.8 ng, respectively. Hypothalamic contents were 24.4% of total in IR-SRIF and 11.4% in IR-NT, respectively. The remainders were found widely in other brain regions. The distribution of IR-SRIF and IR-NT in gastrointestinal tracts revealed the remarkable differences. IR-SRIF was found abundantly in antrum of stomach and pancreas. On the other hand, IR-NT was detected predominantly in jejunum and illeum.
The different distribution of IR-SRIF and IR-NT, especially in gastrointestinal tracts, may be closely related to local physiological roles of these peptides.
