Median Eminence Extracts Research Articles

Properties of CRF from normal and Brattleboro rat median eminence.

Most of the corticotrophin-releasing factor (CRF) activity of normal rat median eminence (ME) extract binds to a neurophysin affinity column. The bound material contains the large and small factors, which we have previously demonstrated to be required together for full activity. Most of the CRF activity of Brattleboro rat ME extract, which contains as much CRF activity as the ME extract of a normal rat, does not bind to a neurophysin affinity column. The CRF activity of Brattleboro rat ME extract resides entirely in a large molecule as determined by Sephadex G-25 chromatography. The different properties of Brattleboro and normal rat CRF suggest that the CRF activity in the Brattleboro rat may result from a substance which is different from that in a normal rat.

Distribution and characterization of corticotropin-releasing factor and arginine vasopressin in rat hypothalamic nuclei.

Corticotropin-releasing factor (CFR) was bioassayed and arginine vasopressin (AVP) radioimmunoassayed from punched-out hypothalamic nuclei. The highest concentration of CFR was found in the median eminence (ME), followed by the paraventricular nucleus (PVN), supraoptic nucleus (SON), suprachiasmatic nucleus (SCN), arcuate nucleus (ARC), dorsomedial nucleus (DMN) and ventromedial nucleus (VMN). The AVP concentration was in the order of ME, SON, PVN, SCN, ARC, VMN and DMN. Sephadex G-25 gel filtration of the ME extracts showed one peak for AVP and two peaks for CRF. One CRF peak appeared on the void volume (big CRF) and the other (small CRF) was coeluted with AVP. Gel filtration of the PVN and SON extracts showed one peak for AVP but three or four peaks for CRF. The addition of anti-AVP serum (AVP-AS) to pituitary cell cultures reduced the CRF activities of AVP and ME extracts by approximately 80 and 40%, respectively. When the small CRF fraction of ME extracts was treated with AVP-AS on affinity chromatography, the unbound fraction (AVP-free) still showed significant CRF activity. Re-examination of CRF concentration using AVP-AS showed that it was still highest in ME, but was significantly higher in PVN than in SON, SCN and ARC. These results suggest that the PVN is an important nucleus for producing corticotropin-releasing hormone.

Effect of Pro-Leu-Gly-NH 2 on plasma levels of α-MSH in the rat

With the idea to give further support that Pro-Leu-Gly-NH 2 (PLG) acts as MIF (the inhibitor factor of MSH), this paper reports the effect of PLG on the secretion of MSH release using a recently developed radioimmunoassay for α-MSH. Pro-Leu-Gly-NH 2 was effective in inhibiting the release of MSH induced by the injection of acid extracts of median eminence (MRF). The rise in plasma MSH by these extracts was not due to their intrinsic content of MSH. Pro-Leu-Gly-NH 2 also inhibited the basal secretion of MSH in male rats. Since PLG also blocked the release of MSH induced by the injection of haloperidol, it is suggested that its effect is not mediated by dopamine.

Stimulation of growth hormone release by vasoactive intestinal polypeptide from human pituitary adenomas in vitro.

The effects of vasoactive intestinal polypeptide (VIP), TRH, dopamine, and rat median eminence extract on GH release from GH-secreting pituitary adenomas were studied in vitro using a sensitive superfusion method. Dispersed pituitary tumor cells obtained from three patients with acromegaly were placed in a superfusion column, and the amounts of GH in the superfusate were determined. The addition of VIP (10(-6) M) to the perfusion system resulted in a marked increase in GH release in all three cases, and a dose-response relationship in VIP (10(-8) 10(-6) M) induced GH secretion was observed in one case studied. TRH (10(-7) M) and median eminence extract (1 equivalent/ml) also caused an abrupt and marked increase in GH release in all of the experiments. The infusion of either dopamine (10(-7) M) or bromocriptine (10(-7) M) inhibited GH secretion. These results suggest that VIP as well as TRH stimulate GH secretion by a direct action on GH-secreting pituitary tumor cells in at least some acromegalic patients.

Morphine and naloxone: effects on beta-endorphin immunoreactivity in canine plasma and secretions from rat pituitaries.

Morphine and naloxone were administered to five dogs to assess their effects on endogenous opioid release. Morphine (3 mg/20 kg) produced a significant (P less than 0.05) increase in plasma beta-endorphin immunoreactivity(beta EI) compared to saline control. The peak stimulation [19.2 +/- 4.97 baseline to 48.1 +/- 6.82 (SEM) pg/ml] occurred at +10 min and rapidly returned to preinjection levels at +60. At a dose 10 times equipotent to circulating basal beta EI, morphine (4-6 micrograms) failed to affect beta EI release. Naloxone, surprisingly, also caused a significant (P less than 0.025) release of beta EI. After naloxone, beta EI rose from a preinjection baseline of 36.4 +/- 5.82 pg/ml to a peak of 172 +/- 44.1 pg/ml at 45 min post injection. Naloxone pretreatment also obscured the effect of subsequently injected morphine (3 mg/20 kg). In three naloxone-treated dogs, gel chromatography of pooled basal and peak plasma revealed a preponderance of beta-lipotropin compared to beta-endorphin. To determine the site of stimulation of beta EI by opiates and opioids, a series of rat anterior pituitary incubations were performed. Neither morphine (10(-6) M) nor D-Ala2-methionine enkephalinamide (10(-6) M) nor naloxone (10(-6) M) had an effect significantly different from control medium on the release of beta EI from the pituitaries. In a second set of experiments we compared the effect on beta EI release of hypothalamic median eminence extract alone or with morphine. Hypothalamic median eminence extract at two concentrations produced significant release of beta EI, which was unaffected by the addition of morphine. These results suggest that stimulation of release of endogenous opioid peptides by opiates occurs at a suprapituitary level.

Ontogenetic development of the inhibition of growth hormone release by somatostatin in the rat: in-vivo and in-vitro (perifusion) study.

An in-vitro study of GH secretion by rat fetal and neonatal pituitary glands was conducted using a perifusion system. After a 2 h period the GH content of the effluent was constant. Theophylline, thyrotrophin releasing hormone (TRH) and rat stalk median emience extract (SME) were effective stimuli of GH release from the pituitary glands of the 19.5-day-old fetuses. Somatostatin, added to the medium (10 microgram/ml), had no inhibitory effect on GH release (basal or stimulated by either theophylline or SME) before day 4 after birth. After postnatal day 5, somatostatin always inhibited GH secretion. These findings were consistent with the results of experiments in vivo. In rats tested within 4 days of birth, sodium pentobarbitone-stimulated plasma GH levels were not reduced by somatostatin; on day 4 and thereafter somatostatin depressed the response to pentobarbitone injection. These results indicate a postnatal maturation of the regulation of GH release by the hypothalamo-hypophysial system in the rat.

Characterization of corticotropin releasing factor (CRF) and arginine vasopressin in median eminence extracts on sephadex gel-filtration.

Hypothalamic median eminence extracts (MEE) were subjected to gel filtration on Sephadex G-25 and G-75 columns to characterize the corticotropin releasing factor (CRF) in relation to arginine vasopressin (AVP). CRF activity was measured using monolayer cultured anterior pituitary cells, and AVP was measured by radioimmunoassay. Sephadex G-25 chromatography of AVP immunoreactivity of MEE (NIAMDD-Rat HE-RP-1) showed three peaks on elution with 0.1 N HCI and four peaks on elution with 0.2M acetic acid. But freshly prepared MEE showed only one peak on elution with 0.1 N HCI. These results suggest that AVP in NIAMDD-Rat HE-RP-1 is polymerized or aggregated on the Sephadex G-25 column, especially when eluted in 0.2M acetic acid. Two main peaks of CRF activity appeared consistently on both Sephadex G-25 and G-75 chromatography. One was near the void volume and the other was retarded. The small molecular CRF (small-CRF) peak was coeluted with immunoreactive AVP and 125I-AVP, on both chromatographies on elution with 0.1 N HCI. The large molecular CRF (big-CRF) appeared between the void volume and I-39 ACTH on Sephadex G-75 chromatography. Big-CRF from freshly prepared MEE had no AVP immunoreactivity. AVP showed CRF activity in pituitary cell cultures, but its CRF activity accounted for no more than 20% of the CRF activity of NIAMDD-Rat HE-RP-1. The log dose-response characteristics of the CRF activities of small-CRF and AVP differed. These results suggest that the median eminence has at least two substances with CRF activity: one is large molecular CRF, and the other is small molecular CRF which may have a vasopressin-like molecular weight. AVP may account for a part of the CRF activity of small molecular CRF but is not identical with genuine CRF.

Concomitant Secretion of Adrenocorticotropin, β-Endorphin, and γ-Melanotropin from Perfused Pituitary Tumor Cells of Cushing's Disease: Effects of Lysine Vasopressin, Rat Median Eminence Extracts, Thyrotropin-Releasing Hormone, and Luteinizing Hormone-Releasing Hormone∗

Regulation of secretion of ACTH-, beta-endorphin-, and gamma-melanotropin-like immunoreactivities (ACTH-LI, beta-EP-LI, and gamma-MSH-LI, respectively) was studied by using a perfused Sephadex column containing dispersed pituitary tumor cells obtained from three patients with Cushing's disease. Serial dilution of the perfusion medium gave lines parallel to the standard curve in each RIA for ACTH, beta-EP and gamma-MSH, suggesting that immunoreactive materials in the medium are immunologically indistinguishable from the authentic peptides. Gel exclusion chromatography of the medium revealed the existence of ACTH, beta-lipotropin (beta-LPH), beta-EP, and their possible precursor protein. gamma-MSH-LI consists of a major peak of big gamma-MSH eluted near the elution position of beta-LPH, suggesting the entire or nearly entire N-terminal portion of the precursor molecule. The addition of lysine vasopressin and rat median eminence extracts (MEE) to the perfusion system concomitantly enhanced the release of ACTH-LI, beta-EP-LI, and gamma-MSH-LI, although the dose-response relationship was clear-cut only in the case of MEE. TRH and LRH also elicited the concomitant release of these peptides in one patient, in whom combined administration of TRH and LRH significantly augmented plasma cortisol levels when studied preoperatively. The molar ratio of ACTH-LI to beta-EP-LI was approximately 1.0, whereas gamma-MSH-LI was about one fourth of ACTH-LI when compared on a weight basis. These results indicate that 1) ACTH-producing human pituitary adenomas concomitantly secrete ACTH, beta-LPH, beta-EP, and big gamma-MSH, and 2) lysine vasopressin, MEE, TRH, and LRH act directly on pituitary cells to stimulate the release of these peptides.

Adrenocorticotropin and lipotropin secretion by dispersed cell cultures of a human corticotropic adenoma: effect of hypothalamic extract, arginine vasopressin, hydrocortisone, and serotonin.

Basal and modulated secretion of ACTH and lipotropin (LPH) by cultures of trypsin-dispersed cells of a biopsy of a human corticotropic adenoma have been examined. ACTH secretion was detectable throughout the period of culture (13 days) but declined steadily from an initial production rate of 238 +/- 124 ng/3 X 10(5) cells/12 h. The time course of secretion showed a slower phase over the first 4 h, with increases up to 12 h. An extract of rat stalk median eminence caused a significant (P less than 0.005) dose-dependent increase in both ACTH and LPH secretion during 30 min. The patterns of response for ACTH and LPH were very similar; both exhibited a decline in the basal release of peptide subsequent to the period of stimulation. The addition of hydrocortisone (0.2 micrograms/ml) did not suppress basal ACTH secretion during 30 min but significantly (P less than 0.05) inhibited stimulation produced by rat stalk median eminence extract. Arginine vasopressin (dose range, 1-9 ng/ml) significantly (P less than 0.025) stimulated both ACTH and LPH secretion during 30 min. The patterns of response were again very similar. Serotonin (dose range, 0.01-10 micrograms/ml) did not affect ACTH secretion during incubations of 30 min to 4 h. The results obtained with the cell cultures of a human corticotropic cell adenoma concur with in vivo findings of incomplete autonomy of secretion, parallel secretion of ACTH and LPH in response to provocative stimuli, and suppression by corticosteroids. The technique has potential for exploring the cellular mechanisms controlling secretion by human corticotropic adenomas as well as the nature of the hormones produced.

Rat posterior lobe modulates growth hormone release [formula omitted] and [formula omitted

Electrical stimulation of hypophysical posterior lobes in vivo evokes a significant decrease of plasma growth hormones (GH) and an increase of plasma corticotropin (ACTH) concentrations. Electrical stimulation of posterior lobes in vitro evokes the simultaneous release of GH inhibiting factor(s) (GHRIF) and ACTH releasing factor(s) (CRF) into the medium. Pretreatment of media with thioglycolate abolishes the CRF and GHRIF activity, but reveals GH releasing factor(s) (GHRF). Median eminence extracts and vasopressin have potent GHRF and CRF activity. Vasopressin may account fully for the CRF and partially for GHRF activity. Results suggest that hypothalamo-neurohypophysical axons release GHRIF, vasopressin and possibly a GHRF into a portal circulation to modulate the secretion of GH and ACTH.

Further evidence for a prolactin-stimulating neurohormone in reptiles

Neutralized acid extract of median eminence from male rats, female turtles ( Pseudemys scripta elegans), and snakes ( Natrix taxispilota) of undetermined sex stimulated the release of prolactin by turtle anterior pituitary explants in organ culture or in short-term incubation. Rat, turtle, or snake cortical extract did not affect prolactin secretion in vitro. The prolactin-releasing activity of both turtle and snake median eminence extract was detectable during the first 4 hr of a 12-hr incubation and showed consistent stimulation throughout the entire incubation period. Both of the reptilian median eminence extracts also enhanced synthesis of prolactin by the turtle anterior pituitary gland in vitro. The results support the hypothesis that the reptilian median eminence contains a factor which stimulates the secretion and synthesis of prolactin and that the prolactin-stimulating factor may be indigenous to the hypothalami of a wider range of reptilian species.

Involvement of vasopressin in corticotropin-releasing effect of hypothalamic median eminence extract.

Incubation of anterior pituitary (AP) fragments of rats was used to determine the specific role played by vasopressin (VP) in the overall effect of crude hypothalamic median eminence (HME) extract on ACTH release. Using the property of an AVP antiserum (AS) to completely abolish the CRF-like effect of hypothalamic VP without apparently affecting the effect of CRF, we show that under specific incubation conditions, the effect of the two secretagogues are additive at the pituitary level. ACTH secretion of pituitaries was enhanced when incubation was carried out in the presence of VP together with a maximum effective dose of VP-free HME extract (from Brattleboro rats). These observations favor the hypothesis that VP and CRF have different receptor sites in the anterior pituitary.

CHANGES IN CORTICOTROPHIN RELEASING FACTOR OF THE STALK MEDIAN EMINENCE IN RATS WITH VARIOUS CUTS AROUND THE MEDIAL BASAL HYPOTHALAMUS

The activity of corticotrophin releasing factor (CRF) in extracts of the stalk median eminence (SME) complex proper (average protein content, 30.6 micrograms) of male rats was assayed by monolayer cultures of anterior pituitary cells using the release of immunoreactive ACTH. Extracts which were equivalent to 0.025 SME of control rats usually had detectable CRF activity, while there was no detectable activity in extracts of 0.4 SME equiv. taken 8 days after complete surgical isolation of the medial basal hypothalamus (MBH). The activity of CRF in extracts from rats with an anterolateral cut around the MBH was at least ten times less than that in the control rats. One day after placing an anterolateral cut around the MBH the ACTH releasing activity of the SME was not significantly different from that of the control animals but activity decreased significantly 3 days after the operation and was at least ten times less than in the control animals on day 7 after the operation. It is suggested that most of the CRF activity of the SME is contained in nerve fibres entering the neurohaemal region from outside the MBH and that transection of these fibres produced the fall in CRF content of the SME in rats with partial or total surgical isolation of the MBH.

Effect of Hypothalamic Extract and Other Factors on Release of Adrenocorticotropin from and Adenosine 3′,5′- Monophosphate Levels in Dispersed Nonpituitary Tumor Cells*

Tumor tissues obtained from two patients with the ectopic ACTH syndrome caused by medullary carcinoma of the thyroid and malignant epithelial thymoma were dispersed by tryptic digestion and mechanical agitation. Using the isolated cells, the effects of various agents on ACTH secretion and intracellular cAMP concentrations were studied. Addition of rat median eminence extract significantly stimulated ACTH secretion and increased levels of intracellular cAMP in both cell preparations, and a dose-response relationship appeared to exist between the dose of rat median eminence extract added and either ACTH secretion or intracellular cAMP formation in the thymic tumor cells. High concentrations of calcium also produced a marked ACTH secretion in both cases. In the thymic tumor cells, norepinephrine, serotonin, and TRH were found to be effective in increasing ACTH secretion and intracellular cAMP levels, whereas biogenic amines, hypothalamic hormones, and gastrointestinal hormones did not affect hormone secretion in the thyroid tumor cells. These results suggest that a corticortropin-releasing factor-like substance(s), as yet unspecified, may play some role in stimulating ectopic ACTH secretion by certain tumors, that both intracellular cAMP and Ca++ may be involved in ectopic hormone secretion, and that the inappropriate hormonal secretory responses of some tumors to a variety of stimuli might be mediated by altered membrane receptors of the neoplastic cells.

Corticotrophin releasing factor may be modulated vasopressin

CORTICOTROPHIN RELEASING FACTOR (CRF) was the first of the postulated hypothalamic factors controlling the release of hormones from the pituitary gland to be demonstrated biologically1. However, it is extraordinary that after 24 years of research, techniques which have established the identity of the hypothalamic peptides involved in the release of thryotrophin, the gonadotrophins and growth hormone, have been unsuccessful when applied to the search for a factor responsible for adrenocorticotrophin (ACTH) release2,3. Vasopressin invariably releases ACTH from the anterior pituitary, but its partial agonistic properties in vitro4–6, its low potency in vivo7 and the isolation of active CRF fractions with little or no vasopressin activity2,3,8,9 have led to its rejection as a physiological CRF10,11. However, recent immunological and immunocytochemical findings have led to renewed interest in the role of vasopressin in ACTH release. High concentrations of immunoreactive vasopressin have been found in portal blood and a direct vasopressin-neurophysin containing axonal pathway to the hypophyseal portal system, which seems to be under the influence of the adrenal cortex, has been identified12,13. Using a sensitive releasing factor bioassay system, the perfused isolated anterior pituitary cell column14, and various immunological techniques, we have presented evidence to support the idea that vasopressin plays a major part in control of ACTH release6, and we now report the existence of hypothalamic factors with weak, labile CRF activity which potentiate the CRF activity of vasopressin to give it full agonistic properties indistinguishable from crude extracts of rat stalk median eminence (SME). Thus we propose that CRF is vasopressin modulated by other hypothalamic factor(s) released into the hypothalamo-hypophyseal portal system.

Characterization of an in vitro method for demonstrating thyrotropin-releasing hormone-stimulated prolactin secretion.

Anterior pituitaries of normal adult male rats were subjected to synthetic thyrotropin-releasing hormone (TRH) treatment in an acute incubation system which employed pretreatment of the glands with plasma obtained from the donor animals. Following a 60-min preincubation period in a 1:1 mixture of Krebs-Ringer bicarbonate buffer (KRB) and plasma, media and hemipituitary prolactin (PRL) concentrations were significantly (p less than 0.01) increased after a 40-min treatment with 500 pg TRH. The TRH effect was absent among hemipituitaries preincubated in KRB alone. Plasma obtained from older donors was more potent than was plasma from younger rats in this effect. TSH secretion was markedly increased by 500 pg TRH, whether or not plasma preincubation was employed. A dose response of PRL release to concentrations of TRH from 100 pg to 6.0 ng was observed. Crude extracts of median eminence also effected enhanced PRL release using the plasma preincubation technique. The results suggest that plasma preincubation of explanted pituitaries increases PRL cell sensitivity to TRH, perhaps by enzymatic inactivation of endogenous TRH bound to cellular membrane receptors.

Characterization of Rat Stalk Median Eminence Vasopressin and Its Involvement in Adrenocorticotropin Release

The perfused isolated rat anterior pituitary cell column was used as a bioassay for corticotropinreleasing factor (CRF). The system responded to arginine vasopressin (AVP) in a dose-dependent manner with a minimum effective dose of 10-10 M. Log doseresponse curves for AVP and crude stalk median eminence extract (SME) were statistically significantly nonparallel (P < 0.05) and similar results were found for lysine vasopressin, arginine vasotocin (AVT), oxytocin, and human posterior lobe extract. Vasopressin and its analogs had no effect on LH release. Using RIA for AVP, SME was found to contain 10.83 ng (7.7–14.3 ng, n = 12) AVP, which could account for no more than 30% of its CRF activity. The CRF activity of both SME and AVP was totally quenched by incubation overnight with vasopressin antisera. Partial quenching was obtained with higher dilutions of the antisera. Vasopressin-stimulated ACTH release was affected in a similar way to SME-stimulated ACTH release by the presence of corticosterone (0.2 μg/ml)...

Perfused Rat Isolated Anterior Pituitary Cell Column as Bioassay for Factor(s) Controlling Release of Adrenocorticotropin: Validation of a Technique

A suspension of isolated rat anterior pituitary cells, with viability of 90% was mixed with 0.5 g preswollen Bio-Gel P2 and packed into a column. ACTH in the fractions collected from the perfused column was measured by immunoassay and bioassay with well correlated results (r = 0.84,P = 0.01). Cells were stimulated with 2–4-min pulses or crude stalk median eminence extract (SME) and responses were recorded as total ACTH released in excess of background. A log doseresponse curve was obtained over the range 0.0025–0.1 SME/ml (one SME = extract of 3–4 mg tissue/ml), giving a highly significant regression of response on dose (P < 0.01) with an index of precision of 0.164 ± 0.007 (mean ± SEM, n = 8). LH, as measured by immunoassay, was also released in response to SME. No significant ACTH release was seen with a cortex extract. ACTH elease was stimulated by 56 mM potassium ions; SMEstimulated ACTH release was dependent on calcium and magnesium ions and was inhibited by the presence of corticosterone (0.2 μg/ml)...

Separation and maturation of gonadotrophs from 2A8 clonal cells in vitro

2A8 clonal cells derived from the epithlium of Rathke's pouch of fetal rats were cultured in growth medium supplemented with fresh rat serum, median-eminence extract and 1-thyroxine. Then, in order to isolate gonadotrophs, cyanogen bromide-activated Sepharose whichwas conjugated with LHRH (LHRH-Sepharose) was added to the culture medium. Fourteen days after incubation of 2A8 cells with LHRH-Sepharose, agranular and granular cells were rapidly bound to LHRH-Sepharose when fresh serum had previously been added in the medium. The cytoplasm of granular cells that were bound to LHRH-Sepharose contained spherical secretory granules (200-250 nm in diameter). These cells were similar in morphology to the FSH and LH gonadotrophs described by Kurosumi (1968). Moreover, many of them appeared as hypertrophied cells that resembled "castration" cells. These results demonstrate that LHRH conjugated to Sepharose can be used to separate gonadotrophs from other 2A8 cells, and it is suggested that the hypertrophy of some cells might be due to persistent stimulation by LHRH which is likely bound to receptors on the cell membrane.

Corticotropin Responsiveness in the Neonatal Rat

Plasma ACTH concentrations were determined in neonatal rats 1, 7, 14, and 21 days old, subjected to treatment with either a median eminence extract with corticotropin releasing factor (CRF) activity or to ether exposure. 'CRF' injection elevated plasma ACTH levels on all days tested. Ether exposure elevated ACTH levels on days 14 and 21, but not on days 1 or 7. These results indicate: (1) that there is no diminution of responsiveness of the pituitary to direct stimulation by CRF as a function of age, and (2) that the mediators of the stress response above the level of the pituitary are not functionally mature until approximately day 14. Correlations with previous studies also indicate that there is a delay in the return of adrenal responsiveness until some time after these mediators attain maturity.