S. M. McCann

LH-Releasing Activity in Hypothalamic Extracts

Summary Acid extracts of rat SME tissue evoked OAAD in immature rats pre-treated with gonadotropins. Part of the activity in the extracts could be accounted for by their content of LH or vasopressin or both, whereas the remaining activity appeared to be due to release of LH from pituitary of assay rats. The substance(s) responsible for LH-releasing activity of extracts has been called LH-releasing factor. The nature of this material is unknown, but it appears to differ from histamine, serotonin, substance P, epinephrine, and vasopressin or oxytocin. The technical assistance cf Maria Smith is gratefully acknowledged.

Evidence for a Role of the Supraopticohypophyseal System in Regulation of Adrenocorticotropic Secretion

Conclusions 1. Hypothalamic lesions which block ACTH secretion, as judged by adrenal ascorbic acid depletion, adrenal weight, or blood ACTH concentration, uniformly destroy a significant fraction of the supraopticohypophyseal tract as evidenced by their location and the presence of diabetes insipidus. 2. ACTH secretion appears to be produced in these rats by large doses of pitressin. 3. Control of diabetes insipidus by small doses of pitressin, or the injection of epinephrine, histamine or pitocin does not produce significant ACTH secretion in such rats. 4. The results indicate that the supraopticohypophyseal tract may play a role in the regulation of ACTH secretion by release of antidiuretic hormone into the hypophyseal portal vessels.

Effect of synthetic vasopressin on release of adrenocorticotrophin in rats with hypothalamic lesions.

Conclusions Commercial and synthetic vasopressin are equipotent in producing ascorbic acid depletion in rats with those hypothalamic lesions which prevent the adrenal response to certain non-specific stimuli. The results are interpreted to mean that vasopressin can evoke ACTH release in these animals and that the ACTH-releasing activity of neurohypophyseal extracts, when tested in vivo, is accounted for by their content of vasopressin. The results support the hypothesis that vasopressin (ADH) is the neurohumor responsible for ACTH release.

Suppression of Adrenal Compensatory Hypertrophy by Hypothalamic Lesions.

Conclusions The compensatory adrenal hypertrophy which normally follows unilateral adrenalectomy is suppressed in rats with diabetes insipidus produced by hypothalamic lesions.

Abundance of Vasopressin and Corticotrophin-Releasing Factor in Neurohypophysial Extracts.∗

Conclusions Extracts of the stalk-median eminence area of beef or rat brain produce a specific release of ACTH in rats with hypothalamic lesions. The major portion of this effect is due to a corticotrophin-releasing factor. Ten to 20% of the activity of these extracts appears to be due to vasopressin.

Blood and pituitary adrenocorticotrophin in adrenalectomized rats with hypothalamic lesions.

Conclusions 1. Blood ACTH concentration of adrenalectomized rats, treated with DCA for two weeks, and then subjected to the acute stress of ether anesthesia and bleeding was found to be 11 mu per 100 ml. 2. Suitably placed hypothalamic lesions prevented this rise in blood ACTH. 3. Pituitary ACTH was maintained at a level approximately 50% of that found in adrenalectomized rats without lesions. 4. The effective lesions interrupted the supraopticohypophyseal tract as evidenced by their location and by the presence of marked diabetes insipidus.

Purification of Ovine Luteinizing Hormone-Releasing Factor by Gel Filtration and Ion Exchange Chromatography.

Summary Acetone powders prepared from sheep SME fragments were extracted with acetic acid and subjected to gel filtration on Sephadex G-25. An LH-releasing zone was eluted from the column just before the appearance of the pressor zone. The LH-releasing zones from 3 such columns were pooled, applied to a CMC column, and eluted by application of gradients of ammonium acetate of increasing ionic strength and pH. A pressor free LH-releasing zone was eluted from the CMC just prior to emergence of vasopressin. This highly purified LH-RF was active at a dose of 3 μg of peptide. Preliminary desalting was required in order for the LH-RF to be retained on the CMC column. Various steps and stages of desalting have been discussed.

Evidence for the Presence of Growth Hormone-Releasing Factor In Blood of Hyperglycemic, Hypophysectomized Rats

Summary The IP or IV injection of 3 ml of plasma from hypoglycemic, hypophysecto-mized rats produced a lowering in hypophyse-al growth hormone (GH) on estimation 30 minutes later by the tibial epiphyseal cartilage test for GH. Plasma from untreated or hypoglycemic, intact rats and from untreated hypophysectomized rats was ineffective. No activity was found in plasma from hypogly-cemic, hypophysectomized rats with hypotha-lamic lesions in the ventral pre-mammillary area. The results are interpreted to mean that hypoglycemia results in the appearance of a circulating GH-releasing factor in the blood of hypophysectomized rats.

Growth Hormone-Releasing Activity of Crude Ovine Hypothalamic Extracts

Summary Crude extracts of ovine SME tissue were effective in depleting pituitary GH of rats at 30 and 60 minutes after injection. Depletion of pituitary GH was estimated by the tibial epiphyseal cartilage test performed in hypophysectomized rats. Injection of extracts into the carotid artery effectively depleted pituitary GH in one of 2 trials, whereas ip or iv injection uniformly evoked GH depletion. There was no apparent difference in the degree of response produced by the 2 latter routes of injection. Extracts were dissolved in several solvents such as physiological saline, 0.1 N acetic acid, or 0.1 M (pH 5.5) ammonium acetate buffer. Extracts dissolved in any of these 3 solvents possessed GH-releasing activity. The ip administration of the acetic acid diluent produced a significant elevation in pituitary GH activity, which is attributed to the irritant effects of the acid. Hypothalamic extract was administered to rats of different sizes in one experiment. GH depletion occurred in rats which weighed either 100 or 200 g, but failed to occur in animals which weighed 500 g. Since the same dose on a weight basis (4 mg/100 g) was given to each group, these results suggest that responsiveness to the GH-releasing factor may decline in older animals. In contrast to the effectiveness of hypothalamic extracts in depleting pituitary GH was the lack of activity of cerebral cortical extracts, synthetic arginine vasopressin, and α melanocyte stimulating hormone. It is concluded that ovine hypothalamus contains a GH-releasing factor.

Purification of hypothalamic corticotrophin-releasing factor (CRF) of ovine origin.

Summary Acetone powders were prepared from large numbers of ovine SME fragments. After preliminary extraction of the powder with glacial acetic acid and lyophilization, the resultant powder was extracted with 0.1 M ammonium acetate and subjected to gel filtration on a long column of Sephadex G-25. A wide zone was eluted from the column which induced adrenal ascorbic acid depletion upon its iv administration into rats with median eminence lesions. Part of this activity was caused by ACTH-like substances, since it was still present in hypophysectomized rats; however, this active zone was free of pressor activity. The active zones from several fractionations with Sephadex were pooled and placed on a CMC column. Elution was accomplished by application of gradients of ammonium acetate buffer of increasing ionic strength and pH. A narrow zone was eluted from the column which was active in inducing adrenal ascorbic acid depletion in rats with lesions, but was inactive in hypophysectomized rats. A dose of < 3 μg of peptide had significant activity. This method appears applicable for preparation of highly purified hypothalamic CRF. In this study we were able to locate only one CRF; however, the possible existence of other CRFs is not excluded.