Michael D. Lumpkin

Intrahypothalamic action of corticotrophin-releasing factor (CRF) to inhibit growth hormone and LH release in the rat

The effects of intravenous or intraventricular injection of synthetic ovine corticotrophin-releasing factor (oCRF) on plasma levels of anterior pituitary hormones were studied in conscious, ovariectomized (OVX) female rats and compared with the actions of the peptide on dispersed anterior pituitary cells from OVX female rats incubated in the presence of CRF. Third ventricular injection of oCRF in freely moving rats caused a significant increase in plasma levels of ACTH in a dose-related manner with a minimal effective dose of less than 0.5 micrograms (0.1 nmol). The effect was observable at 5 min after injection and persisted for the 60 min duration of the experiment. In contrast, growth hormone levels were significantly depressed within 15 min with a minimal effective intraventricular dose of 0.5 micrograms. The suppression persisted for the duration of the experiment but there was no additional effect of the higher dose of 5 micrograms. Plasma LH levels were also lowered by the highest dose of 5 micrograms (1.0 nmol) of oCRF, with the first significant lowering at 30 min. Lower doses had no effect on plasma LH. Plasma TSH levels were not significantly altered. Control injections of the 0.9% NaCl diluent were without effect on the levels of any of the hormones. Intravenous injection of similar doses of oCRF had no effect on plasma levels of GH or LH. The ACTH-releasing action of the oCRF preparation was confirmed by in vitro incubation of the peptide with dispersed anterior pituitary cells for 2 h. A dose-related release of ACTH occurred in doses ranging from 0.1-10 nM, but there were no effects on the release of the other anterior pituitary hormones. The results suggest that oCRF may act within the hypothalamus to suppress the release of GH and to a lesser extent LH. The stimulation of ACTH release following intraventricular CRF is presumably related to its uptake by portal blood vessels with delivery to the pituitary and stimulation of the corticotrophs.

Differential hypothalamic control of FSH secretion: A review

There are many circumstances in which the release of FSH and LH is dissociated; however, many of these are now thought to be brought about by interactions of LH-releasing hormone (LHRH), which stimulates not only LH but also FSH release, and the gonadal peptide, inhibin, which acts at the pituitary to suppress FSH release selectively. There are also many examples which can only be explained by postulating separate hypothalamic control of FSH and LH release. For example, electrochemical stimulation of the medial preoptic area elicited only LH release, whereas stimulation further caudally elicited equivalent LH release but FSH release as well. Points of stimulation particularly in the dorsal anterior hypothalamic area (DAHA) evoked only FSH release. Furthermore, implantation of prostaglandin E2 in various hypothalamic loci in a region extending from the DAHA caudally and ventrally to the caudal median eminence (ME) selectively elicited FSH release. Lesions of the DAHA resulted in a decrease of plasma FSH but not LH in castrated male and female rats and also suppressed the post-castration rise in FSH in males. In ovariectomized estrogen-primed rats with DAHA lesions, injection of progesterone provoked a normal LH surge but a significantly depressed FSH surge. Anterior ME lesions in castrates lowered LH levels more than FSH levels. Extracts of the DAHA evoked greater FSH and LH release in vitro than could be accounted for by the content of LHRH in the extracts, but there was no preferential release of FSH. On the other hand, extracts of the organum vasculosum lamina terminalis (OVLT) evoked dramatically increased FSH release above that which could be accounted for by the content of LHRH. Lastly, posterior ME extracts had more FSH-releasing activity than could be accounted for by their content of LHRH. All these results suggest the existence of an FSH-releasing factor (FSHRF) and lead to the speculation that the cell bodies of FSHRF neurons are located in the DAHA, with axons projecting to the OVLT and to the posterior ME. In other experiments, attempts were made to purify rat and sheep hypothalamic extracts by gel filtration on Sephadex G-25 and to assay the FSH-releasing activity by both bio- and immunoassay. Using this approach, we obtained evidence for the early emergence of a bioactive FSHRF prior to the emergence of LHRH from the column. Although much more work remains to be done, the accumulated evidence strongly supports the concept of a distinct FSHRF.

Motilin: A novel growth hormone releasing agent

Motilin, a gastrointestinal peptide recently detected in the rat brain, was capable of stimulating growth hormone (GH) release from dispersed anterior pituitary cells in a dose-related fashion. In initial experiments, the minimum effective concentration was 10(-7) M and the effect was specific for just GH. Subsequent experiments demonstrated that concentrations of synthetic motilin as low as 10(-9) M could significantly stimulate GH release. Only large IV doses (100 micrograms) of motilin significantly elevated circulating GH levels in vivo. However, administration of antiserum to porcine motilin (100 microliters, IV) significantly depressed plasma GH levels, suggesting a physiologic role for median eminence and hypothalamic motilin in the control of GH secretion. Furthermore, infusion of motilin into the third ventricle of conscious rats resulted in a significant depression of GH levels, suggesting an ultrashort loop feedback action of motilin on the release of motilin itself or somatostatin. In light of motilins only minor structural similarity to human pancreatic tumor GH-releasing factor (GRF) and the ability of passive immunoneutralization of motilin to lower GH, this 22-amino acid peptide must now be considered a physiologic GRF.

Naloxone-induced dissociation of oxytocin and prolactin releases.

Immobilization of adult male rats resulted in concomitant, significant releases of prolactin (PRL) and oxytocin (OT). Naloxone (0.2 mg/kg, i.p.) administration 30 min prior to initiation of restraint resulted in a significant diminution of the PRL response to stress. On the other hand, a significant augmentation of OT release was observed. These results demonstrate that concomitant releases of OT and PRL in response to a given physiologic stimulus (stress) can be dissociated and suggest that the hypothalamic events which are responsible for the release of these hormones during stress are different.

Purification of a bioactive FSH-releasing factor (FSHRF)

Before the advent of radioimmunoassay (RIA), FSH-releasing factor (FSHRF) appeared to be separable from LH-releasing hormone (LHRH) by chromatography followed by bioassay for FSH. In this study, we re-examined hypothalamic extracts for the existence of an FSHRF distinct from LHRH, utilizing the Steelman-Pohley bioassay as well as RIA for identification of FSH. Acid extracts of rat hypothalamic fragments were chromatographed on Sephadex G-25. LH- and FSH-releasing activities of each fraction were assessed by bio- and immunoassay of FSH and immunoassay of LH released after incubation with hemipituitaries from adult male rats. The immunoreactive LHRH(IR-LHRH) concentration of each fraction was also measured by RIA. In order to evaluate the FSH-releasing activity of LHRH, three doses of synthetic LHRH were tested and FSH-releasing activity determined by bio- and immunoassay. By RIA, the FSH-releasing activity of each column fraction could be accounted for by IR-LHRH contamination. However, greater FSH-releasing activity than could be predicted by IR-LRH contamination was detected by Steelman-Pohley assay in fractions eluted prior to the LHRH peak in 2 separate fractionations. These fractions from the second fractionation were pooled and eluted from a CMC column with ammonium acetate buffers. Again greater FSH-releasing activity than could be accounted for by IR-LHRH was detected prior to the IR-LHRH peak by Steelman-Pohley assay. These results agree with early work from our laboratory and suggest the presence of a bioactive FSHRF in hypothalamic extracts.

Presence and possible site of action of secretin in the rat pituitary and hypothalamus

Secretin-like immunoreactivity was detected in extracts of several rat brain structures by radioimmunoassay, most notably in the pituitary, hypothalamus, pineal and septum. Its localization to these structures suggested that it might play a role in neuroendocrine events similar to its structural homolog vasoactive intestinal peptide. Dose-related stimulations (MED, 10(-7) M) of prolactin (PRL) release were observed after incubation of synthetic secretin with dispersed, cultured pituitary cells from male and ovariectomized (OVX) female rats. In OVX females, i.v. infusion of a high dose of secretin (10 micrograms) resulted in a significant elevation of PRL levels. Doses of secretin as low as 0.1 micrograms when administered into the third cerebroventricle were capable of significantly inhibiting PRL release in both males and OVX females, suggesting an ultrashort-loop, negative feedback of secretin. Secretin can now be added to the growing list of putative PRL-releasing agents.

Prolactin-releasing activity of porcine intestinal peptide (PHI-27)

Porcine intestinal peptide (PHI), a twenty-seven amino acid peptide isolated from porcine gut extracts, is a close structural homolog of the secretin family hormones. The structural and biological similarities of PHI to vasoactive intestinal peptide (VIP) together with its presence in the rat hypothalamus suggested a possible role for the peptide in the control of prolactin (PRL) secretion. PHI induced significant, dose-related stimulations of PRL release from cultured, dispersed rat pituitary cells in vitro. The minimum effective dose is 10(-7) molar, compared to 10(-9) molar for VIP. No interactive effect with thyrotropin-releasing hormone was observed; however, PHI partially overcame the dopamine inhibition of PRL release.

Recent studies on the role of brain peptides in control of anterior pituitary hormone secretion.

Recent work in our laboratory on the role of peptides to influence release of pituitary hormones by direct action on the gland and also some of the interactions of these peptides at the hypothalamic level to alter release of pituitary hormones will be reviewed. Considerable evidence from hypothalamic stimulation and lesion studies suggests the existence of a separate FSH-releasing factor (FSHRF). We have been able to purify a bioactive FSHRF which appears to be distinct from LHRH. Consequently, we believe that a distinct FSHRF will ultimately be isolated. With regard to prolactin, it is now clear that it is under dual control by both prolactin-inhibiting (PI) and prolactin-releasing factors (PRF). Although dopamine acts as a PIF, our recent fractionation studies indicate the existence of a peptidic PIF in hypothalamic extracts which can be separated from dopamine and GABA. The peptidic PIF is eluted from Sephadex in the same position originally described by us a number of years ago. Thus, inhibitory control is probably mediated by a combination of factors which would include dopamine, possibly GABA and a peptidic PIF. A number of peptides have been shown to have PRF activity which include TRF and also VIP. In recent studies, we have shown a prolactin-releasing action of oxytocin on male hemipituitaries or dispersed pituitary cells. Furthermore, high doses of oxytocin given intravenously released prolactin in male rats. There is a correlation between estrogen-induced prolactin release and an increase in plasma oxytocin and a correlation between suckling-induced oxytocin and prolactin release. These results suggest that oxytocin may be an important PRF.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for an FSH-releasing factor in the posterior portion of the rat median eminence

To test the hypothesis that an FSH-releasing factor might be contained within the posterior portion of the median eminence (ME), the anterior half of the ME (aME) and the posterior half of the ME (pME) were removed separately from the brains of adult male rats and extracted in 0.2 N acetic acid. LH and FSH-releasing activities of the extracts were measured in vitro by incubating 8 hemipituitaries from adult male rats for 6 h at a dose of 5 tissue equivalents and determining the radioimmunoassayable LH and FSH released into the medium. LH release induced by the aME extracts was significantly greater than that induced by the pME in both experiments, whereas there were no differences in FSH release between aME and pME extracts. A significant dose-related increase in FSH release was noted in this system when 1 and 2 ng of synthetic LHRH were tested which indicates that the assay was sensitive to different amounts of LHRH with regard to FSH-releasing action. The content of immunoreactive LHRH in the extracts was almost twice as high in the aME as in the pME. Therefore, the results indicate that the pME has greater FSH-releasing activity than can be accounted for by its content of LHRH. The additional FSH-releasing activity is presumably due to an FSH-releasing factor distinct from LHRH.

Motilin stimulates growth hormone release in vitro

Motilin, a twenty two amino acid polypeptide originally isolated from duodenal extracts, has been detected recently in the mammalian hypothalamus and pituitary. We have investigated the possibility that motilin might play a role in neuroendocrine events and report here the ability of synthetic porcine motilin (10(-6)M) to stimulate growth hormone release from rat hemipituitaries and dispersed anterior pituitary cells in vitro. No significant effects on luteinizing hormone, thyroid stimulating hormone or prolactin release were observed. Motilin may be added therefore to the growing list of gastrointestinal hormones which can act directly at the level of the anterior pituitary to alter hormone release.