A. M. Lawrence

Hypothalamic prolactin: characterization by radioimmunoassay and bioassay and response to hypophysectomy and restraint stress.

Prompted by immunohistochemical reports of prolactin-like immunoreactivity in cell bodies within the rat hypothalamus, a study was undertaken to quantitate the immunologic and biologic activity of this material. Hypothalamic concentrations of prolactin-like immunoreactivity averaged 402 +/- 23 pg/mg of protein (n = 30). 97% recovery of rat prolactin standards added to homogenates of hypothalamus insured that neuronal tissue, as prepared for these studies, did not interfere with the radioimmunoassay of rat prolactin. Examination of the elution profile from Sephadex G-75 columns of the prolactin-like immunoreactivity in hypothalamic extracts showed that the majority of hypothalamic prolactin-like substance was of a larger molecular size than pituitary prolactin. While increasing amounts of brain extract progressively displaced more I125 prolactin from antibody-binding sites, the displacement curve produced by adding hypothalamic extract was not parallel to that produced by the addition of increasing amounts of anterior pituitary prolactin standards of rat origin. Hypothalamic extracts from hypophysectomized animals, analyzed for biologic activity in the Nb2 lymphoma cell assay, revealed prolactin-like bioactivity, but the bioactivity/immunoactivity (B/I) ratios for hypothalamic extracts were significantly lower than the B/I ratios for pituitary prolactin (0.71 +/- 0.04 for pituitary, vs. 0.19 +/- 0.06 in the hypothalamus; p less than 0.001). 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 the hypothalamus, indicating that brain prolactin-like substance is regulated independently of pituitary prolactin and circulating serum prolactin levels.

TSH in the Rat and Monkey Brain

Characterization of the molecular, immunological and biological properties of a thyrotropin-stimulating hormone (TSH)-like peptide in rodent brain tissue showed similarities to its pituitary counterpart. The distribution of immunoreactivity in rodent brain was determined by radioimmunoassay in both intact and hypophysectomized animals. Easily detectable but smaller quantities of immunoassayable TSH were present in formalin fixed, acetone-stored brains from Macaca mullata. No change in the level of this TSH-like peptide was observed in most rat brain parts, although the hypothalamus did show a significant drop in hormone level after removal of the pituitary. Extracts of brain containing TSH-like material restored thyroid histology to normal when administered to hypophysectomized rats. Tissue cultured neural cells from both intact and hypophysectomized rats released a TSH-like material into the medium over a 30-day time period. Sodium l-thyroxine suppressed TSH release from pituitary monolayers but did not alter TSH release from brain derived cells that were similarly cultured. Surgical thyroidectomy resulted in a striking rise in serum TSH concentrations with no alteration in the content of brain TSH.

Hypothalamic LH May Play a Role in Control of Pituitary LH Release

We have recently reported immunoassayable luteinizing hormone (LH) in several areas of the rat brain and conspicuously present in the hypothalamus. In this report, we focus on the presence of LH in the hypothalamus and its potential role in regulation of pituitary LH release. In adult female rats, examined during the course of the estrous cycle, a significant fall in hypothalamic LH coincides with the surge in pituitary and serum LH at the time of proestrus, signaling ovulation. Ovariectomized adult rats show no change in hypothalamic LH at a time when there is a dramatic rise in both anterior pituitary and serum LH. These data support the concept that hypothalamic LH is not of pituitary origin and that it may play a role in the short-loop negative feedback system controlling the surge in anterior pituitary LH release, and event initiating ovulation.

Immunological Properties of Bovine Proinsulin and Related Fractions

The immunological interrelationships of bovine proinsulin, its related fractions and insulin are described. Proinsulin has been iodinated and the labeled material purified and characterized. Proinsulin and its intermediate fractions, which could be converted to desalanyl insulin by trypsin, reacted more strongly than insulin or the “nonconvertible” insulin-like material with antiserum to purified proinsulin. The order of reactivity was reversed in favor of insulin when an insulin antiserum was used. Preincubation of the antiserum to purified proinsulin with large quantities of insulin allowed the preferential measurement of bovine proinsulin, and the assay was unaffected by further addition of insulin. The isolated C-peptide from proinsulin was effective in displacing I-131-proinsulin from this antibody. Human and porcine proinsulin reacted weakly with antisera to bovine proinsulin. The immunological differences between proinsulins are far greater than among the corresponding insulins, and imply that the C-peptide of proinsulin contains major antigenic determinants. It is probable that specific antisera will be needed for the optimal assay of each species of proinsulin.

Extrahypothalamic brain luteinizing hormone: characterization by radioimmunoassay, chromatography, radioligand assay and bioassay.

We have recently reported that luteinizing hormone (LH) is present in the hypothalamus of rats. It has chromatographic and biologic characteristics similar to pituitary LH. In this report we focus on extrahypothalamic LH that is widely distributed in the rodent central nervous system. This material has a chromatographic profile similar to that of pituitary LH. Serial dilution of this material is parallel with dilutions of rat pituitary LH in the immunoassay. Brain extracts are active in the testis LH radioligand receptor assay and in the rat interstitial cell testosterone secretion bioassay. Prior incubation of extract with LH antibody significantly attenuated both of these activities. Thus, extrahypothalamic LH has immunologic, chromatographic, and biologic characteristics similar to hypothalamic and pituitary LH.

Unexpected parathyroid disease discovered at thyroidectomy in irradiated patients

Eight of 23 patients undergoing total thyroidectomy for radiation-associated nodular thyroid disease were found to have unsuspected parathyroid hyperplasia or adenoma at operation. The total serum calcium level was normal preoperatively in each patient. Serum ionized calcium and parathyroid hormone levels were measured in five patients preoperatively and were normal in each case. These pathologic findings in normocalcemic patients may represent a preclinical form of hyperparathyroidism, which would be further evidence linking radiation to the pathogenesis of hyperparathyroidism. The parathyroid glands should be evaluated both pre-operatively and at operation in all patients who have a history of radiation and require thyroidectomy.

The effect of In Vitro Ethanol Exposure on Basal Growth Hormone Secretion

Suppressive effects of ethanol (ETOH) on in vivo serum growth hormone (GH) levels have been reported in both humans and animals. To determine whether this effect could be mediated directly at the pituitary level, we have designed a series of in vitro experiments utilizing pituitary cells from ETOH naive animals maintained in monolayer culture. We report that ETOH, in doses ranging from 50 to 400 mg%, caused a prompt and sustained reduction in basal GH secretion, as well as a significant fall in intracellular GH content. These data establish that the in vivo effects of ETOH on GH can be accounted for, at least in part, by a direct effect at the pituitary level, possibly due to reduced GH synthesis.

In Vitro Effect of Ethanol Exposure on Basal and GnRH-Stimulated LH Secretion from Pituitary Cells

The question of whether ethanols (ETOHs) known suppressive effect on serum luteinizing hormone (LH) could be mediated directly at the anterior pituitary level was addressed by examining the effects of ETOH in vitro on release of LH from cultured male rat pituitary cells. The impact of added ethanol concentrations ranging from 50 to 400 mg% on LH release was examined in the basal state and after stimulation by gonadotropin-releasing hormone (GnRH) at a dose of 5 x 10(-10) M. While ETOH did not significantly suppress basal LH release, secretion stimulated with GnRH was noted to be attenuated with higher doses of ETOH (greater than or equal to 100 mg%) compared to stimulated control cells. It is concluded that ETOH exposure in vitro alters stimulated LH secretion by acting directly on pituitary gonadotropes.

Impact of endocrine manipulations on brain-based rat growth hormone.

Our laboratory has previously described the widespread distribution of an immunoreactive and bioactive rat growth hormone (rGH)-like protein in rat brain. It has also been demonstrated that regulation of pituitary rGH secretion is at least partly mediated by a short-loop negative feedback system. In such a system, increased levels of rGH, acting at a suprapituitary locus, would decrease pituitary GH secretion. Thus, the present study has dealt with attempts to further investigate the hypothesis that one function of brain-based rGH might be as a mediator of the short-loop negative feedback system controlling pituitary rGH release. If brain-based rGH were to function as a mediator of such a system, then in situations where serum rGH levels are decreased, brain rGH concentrations should increase, indicating activation of a negative feedback loop. In the present communication we report that significantly decreased serum GH levels in oophorectomized and in thyroidectomized rats were coupled with a significant increase in rGH concentrations in the hypothalamus and in the amygdala. By contrast, adrenalectomy, which was not associated with any changes in levels of GH in serum caused no perturbations in levels of rGH in the brain. These discordant changes in serum and brain-based rGH are findings compatible with the hypothesis that one function of brain-based rGH is as a mediator of the short-loop negative feedback system regulating the release of pituitary GH.

The effect of acute ethanol (EtOH) exposure on protein kinase C (PKC) activity in anterior pituitary

Alterations in the protein kinase C (PKC) pathway may interrupt anterior pituitary luteinizing hormone (LH) synthesis and/or secretion, which may impair normal reproductive function. Work by our laboratory and others has shown that EtOH has profound deleterious effects on the regulation of the hypothalamic-pituitary-gonadal (HPG) axis. The present study focuses on PKC translocation from the cytosol to the membrane of anterior pituitary after acute EtOH exposure. Serum levels of LH were measured at three time points (15, 30, and 90 min) after an IP injection of either saline or 3 g/kg EtOH in adult castrated male rats. LH levels dropped significantly (p < 0.03) in EtOH-injected compared to saline-injected control animals. In the same animals, EtOH significantly suppressed PKC localization at its active site at the pituitary cell membrane (p < 0.05). These findings suggest that the mechanism of EtOHs suppression of LH is mediated, at least in part, through a decrease in PKC translocation to the anterior pituitary cell membrane.