Abstract
Although the chemical nature and structure of TRF (Thyrotropin-Releasing Factor) has been discussed and studied for 10 years, it is in not quite clear. In 1969 Folkers had synthesized a tripeptide, pyroglutamylhistidilyproline amide, based on knowledge of chemical nautre obtained earlier and observed distinct TRF activity of this tripeptide.
However, there remained to be studied in details the biological action of synthestic TRF. Since we have received synthestic TRF preparation from Dr. Folker, the studies concerning biological action of synthetic TRF were performed in vivo and in vitro. Three assay systems for detecting TRF activity were adopted in vivo using rat or mice and in vitro using rat pituritay incubation.
1. TRF assay by estimation of rat plasma TSH
Ty-pretreated male rats of Swiss-Webster strain weighing 120-130 g were used. Under pentbarbital anesthesia synthetic TRF was injected into the jugular vein. Blood was collected at 10 and 12 minutes after the injection, and plasma TSH was measured by McKenzie's method. 1.0, 5.0 and 10.0 μg of synthetic TRF induced the most prominent increase of plasma TSH at 10 min. after the injection respectively. A clear dose-response relation was recognized between the doses ranging from 1.0 to 10.0 μg of synthetic TRF, and plasma TSH was found to be increased with each TRF doses.
2. TRF assay by estimation of mouse blood 131I levels.
As reported by Redding, 5μCi of Na 131I and 1.0μCi T4 were administered to mice of D D strain weighing 13-15 g. Blood radioactivity was checked before and 2 hrs. after administration of synthetic TRF on the 3rd day of experiment. In this assay 50 and 100 ng of synthetic TRF increased blood radioactivity.
3. TRF assay in vitro using short term incubation of rat pituitary.
Three halves of rat anterior pituitaries were placed in one flask and incubated by modified Saffran's technique, one flask for the test and the other as control. 1.0 ug of synthetic TRF were added of the incubation medium. A distinct increase of TSH activity in the medium was observed after 60 minutes' incubation. From these findings described above, synthetic TRF (pyroglutamilhistidilproline amide) has a prominent TRF activity both in vivo and in vitro.
Recently a number of neuroendocrinological studies have revealed that releasing factor (RF) plays an essential role in the regulation of pituitary tropic hormone secretion. Though a fastinating problem about chemical structure of TRF has long been obscure, Folkers and his collegues had synthesized a preparation called pyroglutamilhistidilproline amide which has distinct TRF activity both in vivo and in vitro.
Using this synthetic TRF preparation received from Dr. Folkers, prominent effects were observed in rat and mouse as reported before by us. However, it is very important whether these actions of synthtic TRF are specific or not, and also whether there may exist modifying factors of TRF action or not. From the these points of view, the following experiments were performed.
1. Specificity of action of synthetic TRF
10.0 μg of synthetic TRF induced the most prominent increase in rat plasma TSH at 10 minutes after i.v. injection. On the contrary, no TRF effect was observed when 10.0 μg of synthefic TRF was given to the mice directly, showing absence of TSH or TSH like activity in synthetic TRF. TSH effect was found to be not altered when synthtice TRF was administered, mixed with a known amount of beef TSH preparation. These findings may indicate that there is no TSH like activity or potentiation of TSH in synthetic TRF.
2. Influence of thyroxine on synthetic TRF activity.
We studied the pituitary responsivenees to synthetic TRF in T4 pretreated rats. Plasma TSH levels at 10 minutes after injection of synthetic TRF were estimated in rats pretreated with various doses of L-thyroxine Na 4 hrs previously.
