An Assay Method for Corticotropin-Releasing Activity by Intrapituitary Microinjection in the Rat

Persistence of Diurnal Periodicity of CRF Activity in Adrenalectomized and Hypophysectomized Rats

“…Hiroshige et al (1971) did not find the increase of CRF activity in 11 day adrenalectomized rats. On the other hand, Seiden and Brodish (1969) demonstrated that extracts of hypothalami from 6 week adrenallectomized rats contained only half the CRF activity of extracts from intact rats.…”

“…They suggested that a shift of rhythm in ACTH might exist in adrenalectomized rats. Hiroshige et al(1971) found that the peak value of CRF activity showed a shift toward the earlier period of the day, falling at 16:00hr or 2hr earlier than that of normal rats. This phenomenon may represent a shift of this rhythm secondary to the absence of adrenal hormone negative feedback.…”

ACTH content in the anterior pituitary after the infusion of various doses of corticosterone and dexamethasone in normal and adrenalectomized rats.

“…The hypothalamic CRF content was determined using, with slight modifications (Hiroshige et al 1969; Hiroshige and Sakakura 1971), the direct intrapituitary microinjection method of Hiroshige et al (1968). The median eminence (ME) was excised as an oval piece of tissue 2-3 mg in weight and homogenized with ice-cold 0.01 N acetic acid-saline (1 ME/10 µl) and allowed to sit in the cold room for 1 hr.…”

Acth Release by Purified Hypothalamic CRF Preparation as Assayed by Intrapituitary Microinjection

“…In a previous paper (Hiroshige et al, 1968a), we offered a new assay method for CRF activity, i.e. intrapituitary microinjection of test materials into the adenohypophysis, exposed parapharyngeally, of dexamethasoneNembutal blocked rats.…”

“…This method is a modification of the original method of Hedge et al (1966), to observe the direct effect of releasing substances on the anterior pituitary tissue as in the case of in vitro methods, yet to enable the gland respond in situ to test materials, thus avoiding artifacts due to the incubation in artificial media. With this assay method, we have tested in the present experiment ACTH-releasing potency of a puri- (Hiroshige et al, 1968a), the activity was then compared with that of the purified hypothalamic CRF at two dose levels as shown in Figure 2. Two dose factorial analysis showed that the difference in response between the purified CRF and median eminence extract at corresponding dosage levels was not significant and that parallelism was well maintained with a highly significant combined slope (P<0.001).…”