Nicholas V. Emanuele

Growth hormone (GH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH)-like peptides in the rodent brain: non-parallel ontogenetic development with pituitary counterparts

Brain and anterior pituitary growth hormone (GH), thyroid-stimulating hormone (TSH) and luteinizing hormone (LH) were measured during fetal, neonatal, and pubertal life and into adulthood. Immunoassayable GH and TSH could be found in the fetal whole brain before their detection in the fetal pituitary. Developmental patterns of pituitary and brain hormones differed in that pituitary hormones showed a gradual rise in levels from birth to puberty at approximately 20 days of age. Biochemically similar, brain-based peptides demonstrated a remarkable preparturitional surge in concentrations that was limited to a few days immediately preceding birth. Twenty-four hours after birth, brain GH, TSH, and LH had dropped to levels equal to or less than concentrations in the neonatal pituitary and subsequently rose to adult levels around the time of puberty. In these studies it could be shown that both the placental-fetal barrier and the neonatal blood-brain barrier were intact. These observations indicate the presence of two biochemically and immunologically similar but topographically distinct pools of peptides present in the developing brain and in the anterior pituitary gland.

Release of immunoreactive luteinizing hormone (LH) from rat hypothalamus-pars tuberalis explants

A luteinizing hormone (LH)-like molecule has been described in the rodent central nervous system and has been shown to have immunologic, chromatographic, and biologic activity similar to that of pituitary LH. In this communication we report that a depolarizing stimulus, high potassium concentration in the presence of calcium, causes in vitro release of LH from hypothalamic explants containing the pars tuberalis. Release from other brain areas containing LH was not seen nor was release of LH, in vitro, significantly provoked from dorsal segments of the hypothalamus, thus suggesting that most, if not all, of the releasable LH from intact hypothalamic explants may come from cells of the pars tuberalis.

Extrahypothalamic brain prolactin: characterization and evidence for independence from pituitary prolactin

Prompted by reports of immunohistochemical localization of a prolactin-like immunoreactivity (PLI) within the rat brain, a study was undertaken to define the immunologic and biologic characteristics of this material in extrahypothalamic regions of the rat brain. Ninety-seven percent recovery of rat prolactin standard, added to homogenates of brain parts, insured that neuronal tissue did not interfere with the radioimmunoassay for rat prolactin. PLI was consistently found in the cerebellum, thalamus, brainstem (pons-medulla), hippocampus, cerebral cortex and caudate. Examination of the elution profile of each of the extrahypothalamic brain parts from Sephadex G-75 columns showed that, although a small amount of brain PLI elutes in the vicinity of the anterior pituitary prolactin marker, the bulk of brain-based PLI migrates with the void volume and as late eluting, low molecular weight material. While increasing amounts of brain extracts progressively displaced more 125I-prolactin from antibody binding, the displacement curve was not parallel to that produced by the addition of increasing amounts of anterior pituitary prolactin standards of rat origin. Extracts of various brain parts from hypophysectomized animals, analyzed for biologic activity in the Nb2 lymphoma cell assay, revealed prolactin-like bioactivity, but the bioactivity/immunoreactivity ratio for some of the brain parts was significantly lower than that for pituitary prolactin. Hypophysectomy, which led to the expected fall in serum prolactin to undetectable levels, and restraint stress, which resulted in a statistically significant 4-fold rise in serum prolactin, caused no change in prolactin concentrations in extrahypothalamic brain parts, indicating that brain PLI is regulated independently of pituitary prolactin and of circulating serum prolactin levels.

The effect of ethanol on prolactin release from pituitary cells in vitro.

Exposure to ethanol is recognized to cause reproductive impairment in man and animals. Since elevated levels of prolactin will interfere with normal functioning of the hypothalamic-pituitary-gonadal axis, and since ethanol has been shown by others to lead to increased prolactin secretion in vivo, the present in vitro study was undertaken to determine whether there is a direct effect of ethanol (ETOH) on prolactin release. Prolactin release from anterior pituitary cells maintained in monolayer culture and exposed to either no ethanol or media containing ethanol at concentrations of 50, 100, 200, or 400 mg% was measured at 1, 4, 24, 48, 72 hours in incubation. Ethanol added directly to pituitary cells stimulated prolactin release at all time points examined. Significant stimulation occurred with addition of low and mid-range ethanol concentrations (50-200 mg%); no augmented prolactin secretory response was seen with the highest ethanol concentration used (400 mg%). This pattern of response was maintained throughout the entire 72 hour incubation period. Thus, the effect of ethanol on prolactin secretion is mediated, at least in part, at the anterior pituitary level.

Subcellular distribution of hypothalamic prolactin-like immunoreactivity

Prompted by interest in immunohistochemical reports of prolactin-like immunoreactivity (PLI) in the rat hypothalamus, we investigated and have reported that an immunoreactive and bioactive prolactin-like material can be extracted from the rat hypothalamus. In the present communication the subcellular distribution of this protein is reported. Using a sensitive and specific radioimmunoassay for rat prolactin and a standardized procedure for subcellular fractionation of neuronal tissue, we have found that 90% of hypothalamic PLI is particulate-bound with only 10% remaining in the S4 or cytosolic fraction. Almost 80% of the particulate-bound PLI is found in the P2 fraction containing myelin, synaptosomes and mitochondria. When P2 is further fractioned on a discontinuous sucrose density gradient, approximately 66% of the P2-associated PLI was found in subfractions rich in synaptosomes and poor in myelin and mitochondria. Such findings support the probability that hypothalamic PLI functions trans-synaptically as a neuromodulator in the brain.

Failure of in vitro ethanol to inhibit LHRH release from the hypothalamus

The reproductive alterations induced by ethanol (ETOH) in the male rodent have been intensively investigated. Although gonadal effects are well characterized, the impact of ETOH on the hypothalamic peptide luteinizing hormone-releasing hormone (LHRH) has been less well defined. The releasability of hypothalamic LHRH in the presence of ETOH has not been directly studied. We report here that ETOH in concentrations of 50 mg% to 400 mg% failed to inhibit LHRH release in vitro.

The impact of aging on luteinizing hormone (LH) and thyroid-stimulating hormone (TSH) in the rat brain

Immunoreactive and bioactive luteinizing hormone (LH), thyroid-stimulating hormone (TSH) and growth hormone (GH) have been described by this and other laboratories to be present in discrete areas of the rodent and primate brain. In the present studies, LH and TSH concentrations in serum, pituitary, hypothalamus, brainstem, cerebellum, cerebral cortex and hippocampus were measured in male Sprague-Dawley rats at 2.5 months of age, when 11-12 months old and at 24 months of age. There was a significant decrease in hypothalamic TSH concentrations in the 11-12-month-old rats compared to younger animals (672 +/- 127 ng/mg vs 86 +/- 17 ng/mg, P less than 0.001). This was unaccompanied by any changes in TSH levels in serum, anterior pituitary, or in any of the extrahypothalamic brain areas that were examined. In contrast, there was a significant 50% drop in LH concentrations in the anterior pituitary gland of 11-12-month-old animals when compared with young controls (72 +/- 58 micrograms/mg vs 137.2 +/- 27 micrograms/mg, P less than 0.05) without any noted change in serum or brain LH concentrations. Similar decreases in LH concentration were also seen in the anterior pituitary glands of two-year-old animals. These discordant profiles between pituitary and brain LH and TSH provide additional circumstantial evidence that these brain peptides do not represent contaminants from the anterior pituitary. Further, these significant changes in TSH and LH concentrations that develop with aging may implicate these particular peptides in the development of certain features of senescence.

Brain thyroid-stimulating hormone: Effects of endocrine manipulations

We have previously described the presence of and the immunologic, chromatographic and biologic characteristics of a thyroid-stimulating hormone (TSH)-like peptide, widely distributed in the rat and monkey central nervous system (CNS). In order to test the hypothesis that brain TSH, specifically hypothalamic TSH, participates in the turnover of pituitary TSH, we have assessed the effect on hypothalamic TSH of two endocrine manipulations, castration and adrenalectomy, known to significantly decrease pituitary and/or serum TSH in the rat. Orchidectomy led to a significant decline of pituitary and serum TSH while a significant increase in hypothalamic TSH concentrations was seen. Adrenalectomy also led to decreased pituitary concentration of TSH linked with a significant increase in hypothalamic TSH levels. Neither manipulation led to changes in TSH in the extrahypothalamic brain. The inverse relationship wherein serum and/or pituitary TSH decrease is accompanied by an increase in hypothalamic TSH is compatible with a role for hypothalamic TSH in pituitary TSH regulation. The possible significance of these findings in terms of TSH triiodothyronine interactions in the CNS is discussed.