Previous experiments have shown that intraventricular injection of ovine corticotropin (ACTH)-releasing factor (oCRF) in doses too low to elevate plasma ACTH by direct action on the pituitary does not lower plasma ACTH, suggesting that the peptide lacks a negative ultrashortloop feedback action to suppress its own release under resting conditions. The present study was performed to determine whether oCRF has any action to alter CRF release in stress.The peptide was injected into the third ventricle or external jugular vein of freely moving ovariectomized female rats 5 min prior to application of ether stress. When oCRF was injected into the third ventricle in doses of 500 pg (0.1 pmol) or less, there was no significant alteration in plasma ACTH prior to ether stress; however, there was a significantly enhanced increase in plasma ACTH 2 and 5 min after ether stress applied 5 min after intraventricular injection of oCRF at doses of 50 (0.01 pmol) or 150 pg (0.03 pmol). These results suggest that the peptide acts on structures adjacent to the third ventricle to augment stress-induced CRF release. To rule out the possibility that the sensitivity of the pituitary itself to CRF increases dramatically following stress, 10 or 100 ng of oCRF was injected i.v. These doses produced a significant dose-related increase in plasma ACTH at 2, 5, or 15 min. In other groups receiving the same doses of oCRF and ether stressed 5 mI later, plasma ACTH was significantly higher 2 or 5 min after ether stress when compared with plasma ACTH in etherstressed saline-injected animals. However, in contrast with the results of intraventricular injection of oCRF, the release of ACTH was no greater than that obtained by summing the independent effects of exogenous oCRF and the CRF released by stress. We conclude that CRF may have a positive ultrashortloop feedback action to enhance stress-induced ACTH release and that this enhancement is not due to increased sensitivity of anterior pituitary corticotrophs to CRF.Ultrashort-loop feedback of releasing hormones to alter their own release was postulated a number of years ago by Martini and co-workers (1). There now appears to be a number of examples of this type of interaction of peptides within the brain to alter their own release. The existence ofan ultrashortloop feedback mechanism by which somatostatin alters its own release has been suggested (2). Similarly, intraventricular injection of oxytocin appears to inhibit its own release (3).Consequently, we have looked for possible interactions of corticotropin (ACTH)-releasing factor (CRF) with other peptides in the hypothalamus. We found to our surprise that injection of ovine CRF (oCRF) into the third ventricle decreases plasma growth hormone with a minimal effective dose of 0.1 nmol. A higher dose (1.0 nmol) also decreased plasma luteinizing hormone (LH) (4, 5). In both instances, similar results have been obtained following lateral ventricular injection of the peptide (6, 7). Thus, it appears that the CRF neurons interact with those con...

A b s t r a c t . To determine i f LHRH m i g h t a c t w i t h i n t h e b r a i n t o modify i t s own release, repeated blood samples were removed from conscious ovariectomized r a t s and minute doses of LHRH were i n j e c t e d i n t o t h e t h i r d v e n t r i c l e (3V). The e f f e c t of these i n j e ct i o n s on plasma LH and FSH was measured by radioimmunoassay (RIA). The h i g h e r dose o f i n t r a v e n t r i c u l a r LHRH (10 ng i n 2 P I ) induced an increase i n plasma LH w i t h i n 10 min a f t e r i t s i n j e c t i o n . Plasma LH decreased f o r t h e n e x t 60 min. This was followed by r e s t o r a t i o n of LH pulses c h a r a c t e r i s t i c o f t h e ovariectomized r a t . T h i s dose o f LHRH s l i g h t l y e l e v a t e d plasma FSH c o n c e n t r a t i o n s . I n s t a r k cont r a s t , a o f FSH. The r e s u l t s a r e i n t e r p r e t e d t o mean t h a t a t t h e h i g h e r dose, s u f f i c i e n t LHRH reached t h e s i t e o f o r i g i n of t h e hypophyseal p o r t a l vessels i n t h e median eminence so t h a t i t d i f f u s e d i n t o p o r t a l vessels and was d e l i v e r e d t o t h e gonadotrophs t o induce LH r e l e a s e . I n c o n t r a s t , t h e lower dose p r o v i d e d s u f f i c i e n t hypothalamic concentrations of t h e p e p t i d e t o suppress t h e discharge o f t h e LHRH neurons, thereby l e a d i n g t o a d e c l i n e i n plasma LH, i n d i c a t i v e o f an u l t r a s h o r t -l o o p n e g a t i v e feedback o f LHRH t o suppress i t s own r e l e a s e . o 198s s o c i e t y f o r Experimental Biology and Pledicine. We have r e c e n t l y obtained e v idence f o r negative u l t r a s h o r t -l o o p feedback o f some hypothalamic r e l e a s i n g and i n h i b i t i n g hormones t o suppress t h e i r own release. For example, i n j e c t i o n o f somatostatin i n t o t h e 3 r d v e n t r i c l e (3V) e l e v a t e d i nstead of 1 ower i ng growth hormone (GH) r e l e a s e ( 1 ) which i n d i c a t e s t h a t h i g h l o c a l concentrations o f t h e v a t i o n o f GH l e v e l s i n plasma which i n d i c a t e s t h a t GRF e x e r t s a s i m i l a r n e g a t i v e u l t r a s h o r t -l o o p feedback ( 2 ) . The present experiments were designed t o determine i f LHRH e x e r t e d a negat i v e u l t r a s h o r t -l o o p feedback t o i n h i b i t t h e enhanced p u l s a t i l e LH r e l e a s e i n the c a s t r a t e r a t . p e p t i d e i n t h e v i c i n i t y o f t h e ven-M a t e r i a l s and Methods. t r i c l e e i t h e r i n h i b i t e d somatostatin Animals. Female r a t s (180-200 g ) r e l e a s e o r enhanced GH-releasing o f t h e Sprague-Dawl ey s t r a i n f a c t o r (GRF) discharge o r acted by a (Holtzman, Madison, WI) which were combination o f these a c t i o n s t o stimu-housed i n group cages a t a temperature l a t e GH r e l e a s e from t h e adenohypo-of 2 4 9 C w i t h l i g h t s on from physis.S i m i l a r l y , we have demon-0500-1900h were used throughout...

To determine the physiological significance of corticotropin-releasing factor (CRF) in the control of pituitary hormone secretion, highly specific antibodies directed against the peptide were injected either intravenously or intraventricularly (third ventricle) and the effect on plasma levels of pituitary hormones was determined before and after application of ether stress for 1 min. The intravenous injection of CRF antiserum (0.5 ml) did not significantly alter basal corticotropin (ACTH) levels in freely moving ovariectomized rats but largely blocked the increase in plasma ACTH resulting from ether stress. These antibodies had no effect on the etherinduced decline in plasma growth hormone (GH), and they failed to modify plasma luteinizing hormone levels. In a second experiment, CRF antiserum (3 ,Il) or normal rabbit serum was injected into the third ventricle. A blood sample was drawn 24 hr later and immediately thereafter another injection of CRF antiserum or normal rabbit serum was made. There was no modification in the level of any of the hormones 24 hr after the first injections, and they were similar in CRF antiserum and normal rabbit serum-injected animals. After imposition of ether stress, the response of plasma ACTH was nearly completely blocked by the intraventricular CRF antiserum, but the degree of blockade was slightly less than that obtained by intravenous injection. The decline in plasma GH after ether stress was blocked by the intraventricular CRF antiserum.There was no effect of the intraventricular injection of the antiserum on the levels of the other pituitary hormones. The results with intravenous injection of the antisera indicate that CRF plays an extremely important but probably not completely indispensable role in the release of ACTH after ether stress. The results of the intraventricular injection of the antiserum suggest strongly that endogenous CRF may also modify its own release in response to stress, augmenting it by a positive ultrashort loop feedback, and that the antisera against the peptide blocked this action; however, an action at the pituitary of these intraventricularly injected antibodies cannot be completely ruled out. The blockade of the stress-induced suppression of GH release by the CRF antibodies suggests that CRF released intrahypothalamically during ether stress brings about an alteration in the hypothalamic control of GH secretion such that the stress-induced inhibition of GH release is blocked.The control of corticotropin (ACTH) secretion is mediated by corticotropin-releasing factor (CRF) acting in concert with vasopressin and possibly other substances such as oxytocin, epinephrine, and angiotensin 11 (1-3). It has been shown recently that CRF has other actions as well, because intraventricular injection of the peptide resulted in decreased growth hormone (GH) and luteinizing hormone (LH) secretion (4-6). These are hypothalamic actions because there is no effect of this peptide directly on the pituitary to alter secretion of either hormone. The threshold do...

Long-term ovariectomized (OVX) rats were injected in the third cerebral ventricle with 5 ,l~of the globulin fraction of an antiserum raised against a-melanocyte-stimulating hormone (a-MSH) or an equal volume of the globulin fraction of normal rabbit serum (NRS). Immunoneutralization of brain a-MSH produced an increase in the area under the secretion curve of prolactin (Prl), the amplitude of Prl pulses, and mean plasma Prl (P < 0.01). Recently, a-MSH has been found extensively throughout the brain, with high concentrations detected in axons and terminals of the hypophysiotropic area of the hypothalamus (4, 5). The cell bodies containing this peptide have been localized in the arcuate nucleus (5) and more recently in the dorsolateral region of the hypothalamus (6). Based on the distribution of this peptide and its detection in the hypophysial portal blood by a specific radioimmunoassay (RIA) (7), we evaluated the effect of this peptide on the release of anterior pituitary hormones.In recent studies we found that injection of a-MSH into the third ventricle of the brain of ovariectomized (OVX), freely moving rats produced a dose-dependent inhibition of basal and stress-induced release of prolactin (Prl) (8) and inhibited pulsatile release of luteinizing hormone (LH) (9). These effects could be blocked by prior administration of the inhibitor of catecholamine synthesis a-methyl-p-tyrosine or the dopamine receptor blocker spiroperidol (8, 9). a-MSH did not alter Prl and LH release from hemipituitaries and dispersed anterior pituitary cells incubated in vitro, which indicates that this peptide exerts its effect on Prl and LH release via an action in the hypothalamus to stimulate dopamine release, which, in turn, inhibits Prl and LH release (8,9). To determine whether the inhibitory effect of a-MSH may be physiologically relevant in the regulation of these hormones, we passively immunized rats with antisera to a-MSH. MATERIALS AND METHODSAdult female Sprague-Dawley rats of the Holtzman strain were employed. Females were OVX to eliminate the influence of ovarian steroids and elevate gonadotropin levels. The animals were used 3-6 weeks after ovariectomy. Antiserum was delivered to the brain via a cannula implanted in the third brain ventricle (10). A Silastic cannula implanted 24 hr before the experiment in the right external jugular vein (11) was used for withdrawal of blood samples (0.25 ml). After withdrawal of every sample, the blood volume was restored by an injection of an equal volume of heparinized (20 units/ ml), physiological saline. After every four samples, erythrocytes (0.5 ml) prepared previously were injected. The cells were obtained from OVX rats the day prior to the experiment and suspended in 0.9% saline to a hematocrit of 45%.The antiserum used (KDM-1) has been fully characterized (12). Briefly, this antiserum cross-reacts on a molar basis 0.029% with corticotropin (ACTH)-(1-24), 0.004% with ACTH-(1-39), 0.001% with ACTH-(1-10), and 36% with desacetyl-a-MSH and does not cross-react at a 100 mol...

The pattern of alpha MSH release during immobilization stress in ovariectomized rats was determined and correlated with that of plasma PRL and LH. Stress induced a marked elevation in plasma immunoreactive alpha MSH, with a time course identical to that of plasma PRL. The increment in plasma PRL was greater than that in plasma alpha MSH. Plasma LH was markedly lowered by stress. Analysis of pituitary and hypothalamic alpha MSH indicated a significant (P less than 0.05) increase in the neurointermediate lobe and anterior lobe content of alpha MSH. The alpha MSH content in the hypothalamus was lowered by stress when expressed as tissue content (P less than 0.025), although no significant differences in content in this area were detected when the results were expressed in terms of tissue protein. Stress induced a marked increase (P less than 0.01) in the median eminence levels of alpha MSH. Intraventricular (third ventricle) injection of the gamma-globulin fraction of a specific antiserum raised against alpha MSH increased basal PRL levels (P less than 0.025) and prevented the decline in plasma PRL that occurred 60 min after the onset of stress in the normal rabbit serum-injected rats. The stress-induced suppression of plasma LH was attenuated and delayed by the administration of alpha MSH antibodies. In conclusion, alpha MSH of brain origin is released during stress and is involved in lowering plasma PRL to basal levels and producing a partial suppression of plasma LH.